In vivo evaluation of biomimetic fluorosurfactant polymer-coated expanded polytetrafluoroethylene vascular grafts in a porcine carotid artery bypass model.

OBJECTIVE: The objective of this study was to evaluate the potential for biomimetic self-assembling fluorosurfactant polymer (FSP) coatings incorporating heptamaltose (M7-FSP) to block nonspecific protein adsorption, the cell adhesive RGD peptide (RGD-FSP), or the endothelial cell-selective CRRETAWAC peptide (cRRE-FSP) to improve patency and endothelialization in small-diameter expanded polytetrafluoroethylene (ePTFE) vascular graft implants. METHODS: ePTFE vascular grafts (4 mm in diameter, 5 cm in length) were coated with M7-FSP, RGD-FSP, or cRRE-FSP by dissolving FSPs in distilled water and flowing solution through the graft lumen for 24 hours. Coatings were confirmed by receding water contact angle measurements on the lumen surface. RGD-FSP and cRRE-FSP grafts were presodded in vitro with porcine pulmonary artery endothelial cells (PPAECs) using a custom-designed flow system. PPAEC coverage on the lumen surface was visualized with epifluorescent microscopy and quantified. Grafts were implanted as carotid artery interposition bypass grafts in seven pigs for 33 ± 2 days (ePTFE, n = 3; M7-FSP, n = 4; RGD-FSP, n = 3; cRRE-FSP, n = 4). Patency was confirmed immediately after implantation with duplex color flow ultrasound and at explantation with contrast-enhanced angiography. Grafts were sectioned for histology and stained: Movat pentachrome stain to outline vascular layers, immunofluorescent staining to identify endothelial cells (anti-von Willebrand factor antibody), and immunohistochemical staining to identify smooth muscle cells (anti-smooth muscle α-actin antibody). Neointima to lumen area ratio was determined to evaluate neointimal hyperplasia. RESULTS: Receding water contact angle measurements on graft luminal surfaces were significantly lower (P < .05) on FSP-coated ePTFE surfaces (M7-FSP, 40 ± 16 degrees; RGD-FSP, 25 ± 10 degrees; cRRE-FSP, 33 ± 16 degrees) compared with uncoated ePTFE (126 ± 2 degrees), confirming presence of the FSP layer. In vitro sodding of PPAECs on RGD-FSP and cRRE-FSP grafts resulted in a confluent monolayer of PPAECs on the luminal surface, with a similar cell population on RGD-FSP (1200 ± 187 cells/mm(2)) and cRRE-FSP (1134 ± 153 cells/mm(2)) grafts. All grafts were patent immediately after implantation, and one of three uncoated, two of three RGD-FSP, two of four M7-FSP, and two of four cRRE-FSP grafts remained patent after 1 month. PPAEC coverage of the lumen surface was seen in all patent grafts. RGD-FSP grafts had a slightly higher neointima to lumen area ratio (0.53 ± 0.06) compared with uncoated (0.29 ± 0.15), M7-FSP (0.20 ± 0.15), or cRRE-FSP (0.17 ± 0.09) grafts. CONCLUSIONS: Biomimetic FSP-coated ePTFE grafts can be used successfully in vivo and have potential to support endothelialization. Grafts modified with the M7-FSP and cRRE-FSP showed lower intimal hyperplasia compared with RGD-FSP grafts.

Algorithmic approaches to hemostasis testing.

There are many unique issues that may make a pathologist's consultation helpful in hemostasis testing. Besides the rapidly expanding knowledge of both bleeding and thrombotic disorders and a wide test menu, hemostasis testing is very sensitive to preanalytical issues (hemolysis, fill volume, time, temperature, storage conditions) and the interference of many commonly prescribed drugs. The pathologist can serve an important role in the evaluation of a patient for a bleeding or thrombotic disorder. Using defined algorithms, hemostasis testing can proceed in a logical fashion and be reported using patient-specific comments that take into account clinical history and medication therapy. This approach can improve the diagnostic process, preventing misdiagnoses and leading to a decreased time to diagnosis and an improved utilization of laboratory resources.

Evaluation of an automated von Willebrand factor glycoprotein IbM activity assay compared with 3 alternative von Willebrand factor activity assays.

Background: To overcome deficiencies of the traditional von Willebrand factor (VWF) ristocetin cofactor activity assay (VWF:RCo), several automated assays for VWF platelet-binding activity have been developed. Information on the performance of these assays and their diagnostic utility remains limited. Objectives: To validate the VWF:glycoprotein IbM assay INNOVANCE VWF Ac and compare it with an automated VWF:RCo assay as well as with an automated assay and a manual VWF:Ab assay and to generate reference ranges and analyze reproducibility of the VWF:glycoprotein IbM assay. Methods: Clinical sites enrolled healthy subjects and patients representing the intended use population; VWF activity assays were performed, and results were analyzed. The performance of the INNOVANCE VWF Ac assay was also compared between the BCS XP System and the CS-2500 and CS-5100 analyzers. Results: The INNOVANCE VWF Ac assay correlated well with the VWF:RCo assay and the automated HemosIL VWF:Ab assay, with Pearson coefficients of >.9 and a predicted bias of ≤5.0 IU/dL at VWF levels of 30 IU/dL and ≤5.8 IU/dL at the levels of 50 IU/dL, but correlation and bias were not as good when compared with the REAADS manual VWF:Ab assay. Reference ranges observed for healthy subjects correlated well with previously published findings. Reproducibility of the INNOVANCE VWF Ac assay on the BCS XP System and the CS analyzers was excellent, as was correlation among devices. Conclusion: The characteristics of the INNOVANCE VWF Ac assay regarding comparability with other VWF activity assays, reference ranges, and precision support the use of this assay for evaluation of patients with concern for von Willebrand disease.

The relationship between CYP2C19 polymorphisms and ischaemic and bleeding outcomes in stable outpatients: the CHARISMA genetics study.

AIMS: Clinical trials have established the value of clopidogrel therapy in a wide spectrum of patients with cardiovascular diseases. Both loss- and gain-of-function single nucleotide variants of CYP2C19 genes have been identified that affect clopidogrel metabolism and anti-platelet response. We sought to determine the impact of CYP2C19 polymorphisms on ischaemic and bleeding events. METHODS AND RESULTS: A subset of patients from the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial who consented to genotyping was analysed. Patients with clinically evident cardiovascular disease or multiple risk factors were enrolled in the trial. The rates of ischaemic and bleeding events were compared between carriers and non-carriers of loss-of-function and gain-of-function alleles in patients randomized to clopidogrel vs. placebo. A total of 4819 patients were genotyped and available for the analysis. Carriers of CYP2C19 loss-of-function alleles did not have an increased rate of ischaemic events. However, clopidogrel-treated patients did have a significantly lower rate of any bleeding in carriers: 36.1% (240/665) vs. 42.5% (681/1601) in non-carriers, HR: 0.80, 95% CI: 0.69-0.93, P = 0.003 (genotype/treatment interaction, P-value = 0.023). The CYP2C19 gain-of-function alleles did not affect ischaemic or bleeding endpoints. CONCLUSION: No relationship was seen between CYP2C19 status and ischaemic outcomes in stable patients treated with clopidogrel. There was, however, significantly less bleeding with clopidogrel in carriers of the loss-of-function allele, suggesting less anti-platelet response. Although several prior studies, including mainly stented patients, have emphasized the relationship between CYP2C19 loss-of-function alleles and efficacy of clopidogrel, this study of stable patients establishes a potential link with reduced bleeding complications. CLINICAL TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov number, NCT00050817.

Clinical outcomes using a platelet function-guided approach for secondary prevention in patients with ischemic stroke or transient ischemic attack.

BACKGROUND AND PURPOSE: Antiplatelet therapy nonresponse is associated with worse clinical outcomes. We studied the clinical outcomes associated with platelet function-guided modifications in antiplatelet therapy in patients with ischemic stroke or transient ischemic attack. METHODS: From January 2005 to August 2007, 324 patients with ischemic stroke underwent platelet function testing using platelet aggregometry. Aspirin nonresponse was defined as a mean platelet aggregation ≥20% with 0.5 mg/mL arachidonic acid and/or ≥70% with 5 µmol/L adenosine diphosphate. Clopidogrel nonresponse was defined as a mean platelet aggregation ≥40% with 5 µmol/L adenosine diphosphate. A modification was any increase in antiplatelet therapy occurring after testing. Clinical outcomes were compared between patients with and without platelet function-guided antiplatelet therapy modifications using univariate and propensity score-adjusted analyses. RESULTS: In patients with ischemic stroke or transient ischemic attack, 43% (n=128) and 35% (n=54) were nonresponders to aspirin and clopidogrel, respectively. After platelet function testing, antiplatelet therapy was increased in 23% of patients (n=73). After propensity score matching (n=61 in each group), antiplatelet therapy modification was associated with significantly increased rates of death, ischemic events, or bleeding (hazard ratio, 2.24; 95% CI, 1.12-4.47; P=0.02) compared with no modification in antiplatelet therapy and a trend toward increased bleeding (hazard ratio, 3.56; 95% CI, 0.98-12.95; P=0.05). No differences in ischemic events were observed. CONCLUSIONS: Platelet function-guided modification in antiplatelet therapy after an ischemic stroke or transient ischemic attack was associated with significantly higher rates of adverse clinical outcomes.

Design and synthesis of biomimetic hydrogel scaffolds with controlled organization of cyclic RGD peptides.

We report on the rational design and synthesis of a new type of bioactive poly(ethylene glycol) diacrylate (PEGDA) macromers, cyclic Arg-Gly-Asp (cRGD)-PEGDA, to mimic the cell-adhesive properties of extracellular matrix (ECM), aiming to create biomimetic scaffolds with controlled spatial organization of ligands and enhanced cell binding affinity for tissue engineering. To attach the cRGD peptide in the middle of PEGDA chain, a tailed cRGD peptide, c[RGDfE(SSSKK-NH2)] (1), was synthesized with c(RGDfE) linked to a tail of SSSKK. The tail consists of a spacer with three serine residues and a linker with two lysine residues for conjugating with acryloyl-PEG-NHS (5) to create cRGD-PEGDA (6). cRGD-PEGDA possesses good photopolymerization ability to fabricate hydrogel scaffolds under UV radiation. Surface morphology and composition analysis demonstrates that cRGD-PEGDA hydrogels were well-constructed with porous three-dimensional (3D) structures and uniform distribution of cRGD ligands. Our results show that cRGD-PEGDA hydrogels facilitate endothelial cell (EC) adhesion and spreading on the hydrogel surfaces and exhibit significantly higher EC population in comparison with linear RGD-modified hydrogels at low peptide incorporation. Since ligand presentation in biomimetic scaffolds plays an important role in controlling cell behavior, cRGD-PEGDA has great advantages of controlling hydrogel properties and ligand spatial organization in the resulting scaffolds. Furthermore, cRGD-PEGDA is an attractive candidate for the future development of tissue engineering scaffolds with optimum cell adhesive strength and ligand density.

The relation between ABO blood types and clinical and platelet function parameters in patients who underwent percutaneous coronary intervention.

BACKGROUND: ABO blood groups have been associated with venous thromboembolism and arterial thrombosis. Although platelets play key roles in thrombogenesis, the relation between ABO groups and platelets is not well known and was investigated in this study. PATIENTS AND METHODS: ABO blood type information was retrospectively obtained for 206 patients who underwent percutaneous coronary intervention (PCI) and received dual antiplatelet therapy with aspirin and clopidogrel. Platelet function was measured using VerifyNow system, light transmission aggregometry, thromboxane B2, urinary 11-dehydrothromboxane B2, and vasodilator-stimulated phosphoprotein phosphorylation assays. Samples were also tested following treatment with 10 and 30 µmol/l of aspirin or 30 and 100 µmol/l of P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). Forty-four clinical and 30 platelet function parameters were analyzed. Patients were categorized as aspirin or clopidogrel poor responder (PR) according to cutoff levels of each test. RESULTS: Blood type A was significantly associated with myocardial infarction (MI) history [odds ratio (OR)=2.50, 95% confidence interval (CI)=1.37-4.58, P=0.003], higher baseline troponin T and creatine kinase-MB (CK-MB) index, post-PCI CK-MB index, and platelet reactivity index (PRI), and being PR against 2-MeSAMP (OR=5.75, 95% CI=1.51-21.90, P=0.010). Blood type O was associated with higher arachidonic acid-induced platelet aggregation and negatively associated with MI history (OR=0.47, 95% CI=0.26-0.84, P=0.010), PRI and being PR against clopidogrel (OR=0.54, 95% CI=0.30-0.99, P=0.043) and 2-MeSAMP (OR=0.16, 95% CI=0.03-0.76, P=0.021). CONCLUSION: Blood type A was found as a risk factor for MI. Higher arachidonic acid-induced aggregation in group O and higher PRI in group A against aspirin and P2Y12 inhibitor treatment, respectively, may suggest alternative antiplatelet therapies for PRs with these blood types.

Affinity manipulation of surface-conjugated RGD peptide to modulate binding of liposomes to activated platelets.

Platelet adhesion, activation and fibrinogen-mediated aggregation are primary events in vascular thrombosis and occlusion. An injectable delivery system that can carry thrombolytics selectively to the sites of active platelet aggregation has immense potential in minimally invasive targeted therapy of vascular occlusion. To this end we are studying liposomes surface-modified by fibrinogen-mimetic RGD motifs that can selectively target and bind integrin GPIIb-IIIa on activated platelets. Here we report liposome surface-modification with a conformationally constrained high affinity cyclic RGD motif to modulate the GPIIb-IIIa-binding capability of the liposomes. Such affinity enhancement is important for practical in vivo applications to compete with native fibrinogen towards binding GPIIb-IIIa. The platelet-binding of RGD-modified liposomes was studied by fluorescence and scanning electron microscopy, and flow cytometry, in vitro. Binding of RGD-modified liposomes was also tested in vivo in a rat carotid injury model and analyzed ex vivo by fluorescence microscopy. The results from all experiments show that cyclic RGD-liposomes bind activated platelets significantly higher compared to linear RGD-liposomes. Hence, the results establish the feasibility of modulating the platelet-targeting and binding ability of vascularly targeted liposomes by manipulating the affinity of surface-modifying ligands.

Hypersensitivity of platelets to adenosine diphosphate in patients with stable cardiovascular disease predicts major adverse events despite antiplatelet therapy.

Enhanced platelet activity correlates with early markers of myocardial damage in patients with cardiovascular disease. However, the extent to which enhanced platelet function signals subsequent adverse clinical outcomes in patients with cardiovascular disease is unknown. Blood from patients with stable cardiovascular disease receiving aspirin (325 mg/day) as the only antiplatelet therapy was tested for closure time (CT) with the Dade PFA-100 Platelet Function Analyzer system collagen/adenosine diphosphate (ADP) [CADP] cartridge and platelet aggregometry using 10 microM ADP. This study intentionally focused on those patients defined as aspirin sensitive by previously established criteria of arachidonic acid- and ADP-induced platelet aggregometry, and separately by collagen/epinephrine (CEPI) CT using the PFA-100. Follow up averaged 22 months for the adverse clinical events of death, myocardial infarction or cerebrovascular accident. For aspirin sensitivity determined by aggregometry, patients with CADP CT < 90 seconds (125/296 = 42.2%) had a composite endpoint rate of 19.2% (24/125), while those with CADP CT 90 seconds (171/296 = 57.8%) had an endpoint rate of 5.3% (9/171). Patients with CADP CT <90 seconds had a relative risk (RR) of 3.65 (95% CI.: 1.76-7.57) for recurrent events and 6.56 (95% CI.: 1.93-22.35) for death compared to patients with CADP CT 90s. Nearly identical results were obtained when patients were categorized as aspirin sensitive by CEPI CT. Platelet aggregometry with 10 microM ADP yielded no significant RR for the selected outcomes. Platelet function testing using the PFA-100 system appears to identify a subgroup of stable cardiovascular disease patients with increased risk of major adverse events that is associated with hypersensitivity to ADP, regardless of apparently effective aspirin therapy.

The Impact of an Electronic Expensive Test Notification.

OBJECTIVES: The impact of clinical decision support tools (CDSTs) that display test cost information has been variable. METHODS: We retrospectively analyzed the 3-year impact of a passive CDST that notified providers when the test order cost was $1,000 or more. We determined the most common expensive tests ordered, the frequency with which providers abandoned the order after notification, and the costs saved through this intervention. RESULTS: The average monthly abandonment rate was 12.5% (2014), 12.9% (2015), and 14.3% (2016). The cost savings from tests not performed for this 3-year period was $696,007. Molecular hematopathology assays were the most frequently ordered tests, with variable abandonment rates. CONCLUSIONS: Although this CDST was passive (ie, could be overridden at the point of order entry) and was associated with a relatively low abandonment rate, it achieved a considerable cost savings each year since each abandoned test saved the institution $1,000 or more.

Establishing a Clinical Laboratory in a Tertiary/Quaternary Care Greenfield Hospital in the Middle East: Recounting the Cleveland Clinic Abu Dhabi Experience.

CONTEXT: - This review chronicles the establishment of a clinical laboratory in Cleveland Clinic Abu Dhabi, a greenfield tertiary/quaternary care hospital in the United Arab Emirates. It discusses the challenges faced, solutions sought, and lessons learned and shares insights and pitfalls that may be encountered in such an undertaking. OBJECTIVES: - To share our experience in building a clinical laboratory in a start-up, multispecialty hospital and how we provided support and managed people, processes, and technology for building and making operational the Cleveland Clinic Abu Dhabi. DATA SOURCES: - The Medline (PubMed, National Center for Biotechnology Information, Bethesda, Maryland) database was used to review this topic as well as other journals, books, and Google (Mountain View, California) search engine. CONCLUSIONS: - To deliver on the promise of quality healthcare in a culturally appropriate setting close to home, Cleveland Clinic Abu Dhabi proved to be an unprecedented and ambitious project, jointly carried out by Mubadala Investment Corporation and the Cleveland Clinic Foundation. Cognizant of the scale of this task, hospital leadership engaged closely with staff and stakeholders through motivational techniques and effective communication. Excellent project planning and execution of complex tasks were required for initiation of services. Establishing the clinical laboratory served as an instructive model in fostering multidisciplinary teamwork by highlighting ways to manage operational roadblocks and opportunities in the planning, commissioning, and activation phases. Throughout the activation process, all efforts were directed to create a patient-safety culture within an intentional-learning organization.

A biomimetic peptide fluorosurfactant polymer for endothelialization of ePTFE with limited platelet adhesion.

Endothelialization of expanded polytetrafluoroethylene (ePTFE) has the potential to improve long-term patency for small-diameter vascular grafts. Successful endothelialization requires ePTFE surface modification to permit cell attachment to this otherwise non-adhesive substrate. We report here on a peptide fluorosurfactant polymer (FSP) biomimetic construct that promotes endothelial cell (EC)-selective attachment, growth, shear stability, and function on ePTFE. The peptide FSP consists of a flexible poly(vinyl amine) backbone with EC-selective peptide ligands for specific cell adhesion and pendant fluorocarbon branches for stable anchorage to underlying ePTFE. The EC-selective peptide (primary sequence: Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys, CRRETAWAC) has demonstrated high binding affinity for the alpha(5)beta(1) integrin found on ECs. Here, we demonstrate low affinity of CRRETAWAC for platelets and platelet integrins, thus providing it with EC-selectivity. This EC-selectivity could potentially facilitate rapid in vivo endothelialization and healing without thrombosis for small-diameter ePTFE vascular grafts.

Lack of effect of enteric coating on aspirin-induced inhibition of platelet aggregation in healthy volunteers.

BACKGROUND: Aspirin inhibits platelet aggregation and is widely used in the treatment of cardiovascular disease. Some individuals are less responsive to aspirin's antiplatelet effect, a phenomenon termed aspirin resistance. It is not known whether the antiplatelet effect is fully preserved with the enteric-coated (EC) formulation. METHODS: We performed a prospective randomized trial of 50 healthy volunteers using a crossover design to compare the EC with the standard aspirin formulations. The subjects received a 7-day course of each aspirin formulation (81-mg) (Bayer Corporation, Morristown, NJ) separated by a 3-week washout period. Platelet function was measured before and after each course using optical aggregometry (with arachidonic acid and adenosine diphosphate as agonists) and a point-of-care platelet assay. RESULTS: The assays were reproducible, and the variation in baseline platelet function was small to moderate between the subjects. There was no difference in the extent of platelet inhibition between the EC and standard formulations with any of the 3 assays. With the point-of-care platelet assay, the mean aspirin effect favoring the standard formulation (more aggregation inhibition) compared with the EC formulation was 1.6% +/- 15.8% (P = .60 for difference between the formulations). The corresponding optical aggregometry values were -3.4% +/- 39.5% (P = .97) and -1.4% +/- 16.6% (P = .75) for arachidonic acid and adenosine diphosphate, respectively. CONCLUSIONS: Compared with standard aspirin, EC aspirin appears to exhibit similar inhibition of platelet aggregation in healthy volunteers. Furthermore, point-of-care platelet assessment correlated well with the gold standard of laboratory-based optical platelet aggregometry.

The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.

Dual biofunctional polymer modifications to address endothelialization and smooth muscle cell integration of ePTFE vascular grafts.

Expanded polytetrafluoroethylene (ePTFE) grafts were coated on the luminal surface with a cell-adhesive fluorosurfactant (FSP) polymer to promote endothelialization, followed by ethanol hydration to degas the pores and subsequent cell-adhesive, enzymatically degradable poly(ethylene glycol)-based hydrogel incorporation into the graft interstices to accommodate potential smooth muscle cell integration in the graft wall. The FSP coating on ePTFE was stable as demonstrated by a significantly reduced receding water contact angle on FSP-coated ePTFE (14.5 ± 6.4°) compared to uncoated ePTFE (105.3 ± 4.5°, P < 0.05) after ethanol exposure. X-ray photoelectron spectroscopy analysis of the same surfaces confirmed FSP presence. Localization of the FSP and hydrogel within the ePTFE graft construct was assessed using fluorescently labeled polymers, and demonstrated hydrogel infiltration throughout the thickness of the graft wall, with FSP coating limited to the lumen and adventitial surfaces. FSP at the luminal surface on dual-coated grafts was able to bind endothelial cells (EC) (98.7 ± 23.1 cells/mm(2) ) similar to fibronectin controls (129.4 ± 40.7 cells/mm(2) ), and significantly higher than uncoated ePTFE (31.6 ± 19 cells/mm(2,) P < 0.05). These results indicate that ePTFE grafts can be simultaneously modified with two different polymers that have potential to directly address both endothelialization and intimal hyperplasia. Such a construct is a promising candidate for an off-the-shelf synthetic graft for small-diameter graft applications.

Fosinopril versus amlodipine comparative treatments study: a randomized trial to assess effects on plasminogen activator inhibitor-1.

BACKGROUND: ACE inhibitors and calcium antagonists may modulate fibrinolysis. We conducted a randomized controlled trial to assess the effects of these drugs on plasminogen activator inhibitor-1 (PAI-1) antigen, an inhibitor of fibrinolysis. METHODS AND RESULTS: Participants with hypertension and type 2 diabetes mellitus (n=96, 51% black) were randomized after an initial 4 weeks of placebo to double-blind 20 or 40 mg fosinopril or 5 or 10 mg amlodipine daily for 4 weeks in a fixed-dose regimen. After 4 weeks of placebo washout, the patients received 4 weeks of crossover treatments. After treatment with placebo, systolic and diastolic blood pressure were 143+/-2 and 86+/-1 mm Hg and plasma PAI-1 was 43.4+/-2.3 ng/mL. Amlodipine achieved a greater systolic and diastolic blood pressure reduction than fosinopril (10 mm Hg versus 8 mm Hg, P=0.029, and 5 mm Hg versus 3 mm Hg, P=0.040, respectively) but tended to increase PAI-1, whereas fosinopril tended to decrease PAI-1 (5.4+/-3.6 versus -3.8+/-2.5 ng/mL, P=0.045). The PAI-1 changes depended on drug dose (6.5+/-6.1 and 3.4+/-3.9 ng/mL with amlodipine 10 and 5 mg, respectively, and -0.4+/-3.1 and -7.4+/-4.0 ng/mL with fosinopril 20 and 40 mg, respectively, P for trend 0.024). No significant differences between fosinopril and amlodipine were found for short-term changes in tissue plasminogen activator antigen, fibrinogen, C-reactive protein, and interleukin-6. The findings were similar in black and white participants. CONCLUSIONS: Short-term treatment with fosinopril significantly reduced PAI-1 compared with amlodipine in a dose-dependent fashion. This effect, which was independent of blood pressure reduction, may account for the improved clinical outcomes achieved with ACE inhibitors compared with calcium antagonists.

A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease.

OBJECTIVES: This study was designed to determine if aspirin resistance is associated with clinical events. BACKGROUND: Aspirin resistance, defined by platelet function testing and presumed clinical unresponsiveness to aspirin, has been previously reported by our group and others. However, little information exists linking the laboratory documentation of aspirin resistance and long-term clinical events. METHODS: We prospectively enrolled 326 stable cardiovascular patients from 1997 to 1999 on aspirin (325 mg/day for > or =7 days) and no other antiplatelet agents. We tested for aspirin sensitivity by optical platelet aggregation using adenosine diphosphate (ADP) and arachidonic acid (AA). The primary outcome was the composite of death, myocardial infarction (MI), or cerebrovascular accident (CVA). Mean follow-up was 679 +/- 185 days. Aspirin resistance was defined as a mean aggregation of > or =70% with 10 microM ADP and > or =20% with 0.5 mg/ml AA. RESULTS: Of the patients studied, 17 (5.2%) were aspirin resistant and 309 (94.8%) were not aspirin resistant. During follow-up, aspirin resistance was associated with an increased risk of death, MI, or CVA compared with patients who were aspirin sensitive (24% vs. 10%, hazard ratio [HR] 3.12, 95% confidence interval [CI] 1.10 to 8.90, p = 0.03). Stratified multivariate analyses identified platelet count, age, heart failure, and aspirin resistance to be independently associated with major adverse long-term outcomes (HR for aspirin resistance 4.14, 95% CI 1.42 to 12.06, p = 0.009). CONCLUSIONS: This study demonstrates the natural history of aspirin resistance in a stable population, documenting a greater than threefold increase in the risk of major adverse events associated with aspirin resistance.

RGD-modified liposomes targeted to activated platelets as a potential vascular drug delivery system.

Local drug delivery has become an important treatment modality for the prevention of thrombotic events following coronary angioplasty. In this study, we investigate the ability of liposomes bearing surface conjugated linear Arg-Gly-Asp (RGD) peptide (GSSSGRGDSPA) moieties to target and bind activated platelets, and the effect of such RGD-modified liposomes on platelet activation and aggregation. The binding of RGD-liposomes to human platelets was assessed by fluorescence microscopy, phase contrast microscopy and flow cytometry. The effect of RGD-modified liposomes on platelet activation and aggregation was investigated in vitro, with and without platelet agonists. RGD-liposomes were found to bind activated platelets at levels significantly greater than the control RGE-liposomes. The RGD-liposomes did not exhibit any statistically significant effect on platelet activation or aggregation. The results demonstrate the ability of the RGD-modified liposomes to target and bind activated platelets without causing significant platelet aggregation and suggests a feasible way for the development of a platelet-targeted anti-thrombogenic drug delivery system. Furthermore, the approach can be extended to the development of liposomes for other vascular targets, for application in drug delivery or gene therapy.