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Background The safety and efficacy of edoxaban and dalteparin is unclear for several cancer groups. Methods We evaluated the occurrence of the primary outcome in large cancer groups. The primary outcome was the composite of recurrent VTE or major bleeding over 12 months. Results In patients with gastrointestinal cancer, the primary outcome occurred in 19.4% patients given edoxaban and in 15.0% given dalteparin (risk difference [RD], 4.4%; 95%-CI, −4.1% to 12.8%). The corresponding rates for edoxaban and dalteparin were 10.4% and 10.7% for lung cancer (RD, −0.3%; 95%-CI, −10.0% to 9.5%), 13.6% and 12.5% for urogenital cancer (RD, 1.1; 95%-CI, −10.1–12.4), 3.1% and 11.7% for breast cancer (RD, −8.6; 95%-CI, −19.3–2.2), 8.9% and 10.9% for hematological malignancies (RD, −2.0; 95%-CI, −13.1–9.1), and 10.4% and 17.4% for gynecological cancer (RD, −7.0; 95%-CI, −19.8–5.7). In the subgroup of gastrointestinal cancer, edoxaban was associated with a 3.5% lower absolute risk of recurrent VTE and a 7.9% higher risk of major bleeding. Conclusion Edoxaban has a similar risk-benefit ratio to dalteparin in most cancer groups. In those with gastrointestinal cancer, the lower risk of recurrent VTE and the advantages of oral therapy need to be balanced against the increased risk of major bleeding.

BackgroundIn the ORAL Surveillance study of patients (pts) aged ≥50 yrs with moderate to severe rheumatoid arthritis (RA) and ≥1 additional cardiovascular risk factor (NCT02092467), the incidence of pulmonary embolism was higher with tofacitinib than with tumour necrosis factor inhibitors (TNFi).1ObjectivesTo explore whether biomarkers explained the associations of tofacitinib vs TNFi with venous thromboembolism (VTE) in ORAL Surveillance.MethodsORAL Surveillance was a prospective, open-label, event-driven, noninferiority, post-authorisation safety study. Pts were randomised 1:1:1 to receive tofacitinib 5 or 10 mg twice daily or a TNFi (adalimumab 40 mg every 2 weeks or etanercept 50 mg once weekly). For this exploratory post hoc analysis, 294 soluble, proteomic, genetic and antibody biomarkers were assessed (of which 79 have a known role in inflammation, coagulation, vascular biology and/or Janus kinase signalling). Biomarkers were quantified in serum collected at baseline (BL) and Month (M)12 in VTE cases and 4:1 matched controls. D-dimer was analysed with a larger control group (all eligible pts without VTE) and final adjudicated data from BL, M12 and study end.ResultsOf the 4362 randomised and treated pts, D-dimer was quantified in 3732 pts (54 with VTE; 3678 without) and the remaining biomarkers were analysed in 285 pts (57 VTE cases; 228 matched controls). BL characteristics were generally similar in those with or without VTE and between treatment groups. At BL, D-dimer levels were ≥2×upper limit of normal for ~50% of controls and 67% of VTE cases. Mean D-dimer levels decreased from BL to M12 in controls across treatment groups (Figure 1). Key findings from the biomarker analyses are shown in the Table 1. No biomarker showed a clear mechanistic association with the increased risk of VTE for tofacitinib vs TNFi, or demonstrated adequate performance for prognostic use in pts with RA.Table 1.Summary of results from biomarker analysesBiomarkerKey resultsTier 1C-reactive protein•No association with VTE in any treatment arm at BL or M12D-dimer Thrombopoietin•Higher M12 levels were prospectively associated with greater risk of subsequent VTE with tofacitinib 10 mg BID ◦ For D-dimer, the same effect was observed with tofacitinib 5 mg BID •Treatment specificity of effects could not be establishedTier 2Factor VIII Thrombin–antithrombin complex Tissue factor pathway inhibitor Plasminogen activator inhibitor-1 Protein C Antithrombin Apolipoprotein C-III Leptin•No clinically meaningful differences across treatment armsTiers 3 & 4Exploratory proteomic assays (276 markers from multiplex panels)•Two biomarkers with no known relationship to VTE (angiogenin and TNFSF13B) showed significant associations with pulmonary embolism in the tofacitinib 10 mg BID arm ◦ Treatment specificity of effects could not be established for either analyteGenetic biomarkersFactor V Leiden R506Q, prothrombin G20210A and JAK2 V617F mutations•Factor V Leiden and prothrombin risk alleles, individually or combined, were associated with increased incidence of VTE but did not explain excess events with tofacitinib •No VTE cases or matched controls had the JAK2 mutationAntibody biomarkersACA IgG and IgM, anti-β2GP1 IgG and IgM•No statistical differences were observed between treatment arms or between VTE cases and matched controlsACA, anticardiolipin antibody; β2GP1, beta-2-glycoprotein 1; IgG, immunoglobulin G; IgM, immunoglobulin M; JAK2, Janus kinase 2; TNFSF13B, tumour necrosis factor ligand superfamily member 13BConclusionThis post hoc exploratory analysis did not identify biomarkers at BL or M12 that explain the increased VTE risk for tofacitinib vs TNFi. Notably, ORAL Surveillance was neither designed nor powered to compare the risk of VTE across treatments or to identify biomarkers with a mechanistic relationship to VTE. Individual VTE risk should be considered when making decisions about initiation or maintenance of tofacitinib treatment.References[1]Ytterberg et al. N Engl J Med 2022; 386: 316-326.AcknowledgementsStudy sponsored by Pfizer Inc. Medical writing support was provided by Julia King, CMC Connect, and funded by Pfizer Inc.Disclosure of InterestsZoltán Szekanecz Speakers bureau: AbbVie, Eli Lilly, Novartis, Pfizer Inc, Roche and Sanofi, Paid instructor for: AbbVie, Eli Lilly, Gedeon Richter, Novartis, Pfizer Inc and Roche, Consultant of: AbbVie, Eli Lilly, Novartis, Pfizer Inc, Roche and Sanofi, Grant/research support from: Pfizer Inc, Christina Charles-Schoeman Consultant of: AbbVie, Gilead Sciences, Pfizer Inc and Sanofi-Regeneron, Grant/research support from: AbbVie, Bristol-Myers Squibb and Pfizer Inc, Ivana Vranic Shareholder of: Pfizer Inc, Employee of: Pfizer Ltd, Burak Sahin Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Sara A Paciga Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Zhenyu Wang Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Craig Hyde Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, David Martin Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Jeffrey I Weitz Speakers bureau: Anthos, Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Ionis, Janssen, Novartis, Pfizer Inc, PhaseBio, Portola and Servier Pharmaceuticals, Grant/research support from: Bayer AG and Boehringer Ingelheim

The authors regret that panel E of Fig. 1 was missing, showing the cumulative incidence of the primary outcome in hematologic cancer. The authors would like to apologise for any inconvenience caused.

Essentials Coronary artery bypass graft (CABG) failure is associated with myocardial infarction and death. We tested whether more frequent dosing improves aspirin (ASA) response following CABG surgery. Twice-daily compared with once-daily dosing reduces ASA hyporesponsiveness after CABG surgery. The efficacy of twice-daily ASA needs to be tested in a trial powered for clinical outcomes. SummaryBackground Acetyl-salicylic acid (ASA) hyporesponsiveness occurs transiently after coronary artery bypass graft (CABG) surgery and may compromise the effectiveness of ASA in reducing thrombotic graft failure. A reduced response to ASA 81 mg once-daily after CABG surgery is overcome by four times daily ASA dosing. Objectives To determine whether ASA 325 mg once-daily or 162 mg twice-daily overcomes a reduced response to ASA 81 mg once-daily after CABG surgery. Methods Adults undergoing CABG surgery were randomized to ASA 81 mg once-daily, 325 mg once-daily or 162 mg twice-daily. The primary outcome was median serum thromboxane B2 (TXB2) level on postoperative day 4. We pooled the results with those of our earlier study to obtain better estimates of the effect of ASA 325 mg once-daily or in divided doses over 24 h. Results We randomized 68 patients undergoing CABG surgery. On postoperative day 4, patients randomized to receive ASA 81 mg once-daily had a median day 4 TXB2 level of 4.2 ng mL−1 (Q1, Q3: 1.5, 7.5 ng mL−1), which was higher than in those randomized to ASA 162 mg twice-daily (1.1 ng mL−1; Q1, Q3: 0.7, 2.7 ng mL−1) and similar to those randomized to ASA 325 mg once-daily (1.9 ng mL−1; Q1, Q3: 0.9, 4.7 ng mL−1). Pooled data showed that the median TXB2 level on day 4 in groups receiving ASA 162 mg twice-daily or 81 mg four times daily was 1.1 ng mL−1 compared with 2.2 ng mL−1 in those receiving ASA 325 mg once-daily. Conclusions Multiple daily dosing of ASA is more effective than ASA 81 mg once-daily or 325 mg once-daily at suppressing serum TXB2 formation after CABG surgery. A twice-daily treatment regimen needs to be tested in a clinical outcome study.

ESSENTIALS: It is not known if D-dimer testing alone can safely exclude pulmonary embolism (PE). We studied the safety of using a quantitative latex agglutination D-dimer to exclude PE in 808 patients. 52% of patients with suspected PE had a negative D-dimer test and were followed for 3 months. The negative predictive value of D-dimer testing alone was 99.8%, suggesting it may safely exclude PE. Background Strategies are needed to exclude pulmonary embolism (PE) efficiently without the need for imaging tests. Although validated rules for clinical probability assessment can be combined with D-dimer testing to safely exclude PE, the rules can be complicated or partially subjective, which limits their use. Objectives To determine if PE can be safely excluded in patients with a negative D-dimer without incorporating clinical probability assessment. Patients/methods We enrolled consecutive outpatients and inpatients with suspected PE from four tertiary care hospitals. All patients underwent D-dimer testing using the MDA D-dimer test, a quantitative latex agglutination assay. PE was excluded in patients with a D-dimer less than 750 μg FEU L(-1) without further testing. Patients with D-dimer levels of 750 μg FEU L(-1) or higher underwent standardized imaging tests for PE. All patients in whom PE was excluded had anticoagulant therapy withheld and were followed for 3 months for venous thromboembolism (VTE). Suspected events during follow-up were adjudicated centrally. Results Eight hundred and eight patients were enrolled, of whom 99 (12%) were diagnosed with VTE at presentation. Four hundred and twenty (52%) patients had a negative D-dimer level at presentation and were not treated with anticoagulants; of these, one had VTE during follow-up. The negative predictive value of D-dimer testing for PE was 99.8% (95% confidence interval, 98.7-99.9%). Conclusions A negative latex agglutination D-dimer assay is seen in about one-half of patients with suspected PE and reliably excludes PE as a stand-alone test.

Blood-contacting medical devices, such as vascular grafts, stents, heart valves, and catheters, are often used to treat cardiovascular diseases. Thrombus formation is a common cause of failure of these devices. This study (i) examines the interface between devices and blood, (ii) reviews the pathogenesis of clotting on blood-contacting medical devices, (iii) describes contemporary methods to prevent thrombosis on blood-contacting medical devices, (iv) explains why some anticoagulants are better than others for prevention of thrombosis on medical devices, and (v) identifies future directions in biomaterial research for prevention of thrombosis on blood-contacting medical devices.

Summary Background The efficacy of ASA for prevention of graft failure following CABG surgery may be limited by incomplete platelet inhibition due to increased post-operative platelet turnover. Objectives To determine whether acetyl-salicylic acid (ASA) 325 mg once-daily or 81 mg four-times daily overcomes the impaired response to ASA 81 mg once-daily in post-operative coronary artery bypass graft (CABG) patients. Methods We randomized 110 patients undergoing CABG surgery to either ASA 81 mg once-daily, 81 mg four times daily or 325 mg once-daily and compared their effects on serum thromboxane B2 (TXB2) suppression and arachidonate-induced platelet aggregation. Results One hundred patients were included in the final analysis. Platelet counts fell after surgery, reached a nadir on day 2, and then gradually increased. Although there was near complete suppression of TXB2 on the second or third post-operative day, TXB2 levels increased in parallel with the rise in platelet count on subsequent days. This increase was most marked in patients receiving ASA 81 mg once-daily and less evident in those receiving ASA four times daily. On post-operative day 4, (i) median TXB2 levels were lower with four times daily ASA than with either ASA 81 mg once-daily (1.1 ng/mL; Quartile(Q) Q1,Q3: 0.5, 2.4 and 13.3 ng/mL; Q1,Q3: 7.8, 30.8 ng/mL, respectively; P

Essentials Specific reversal agents for managing severe factor Xa inhibitor-associated bleeding are lacking. We assessed 4-factor-prothrombin complex concentrate (4F-PCC) and tranexamic acid (TXA). 4F-PCC, but not TXA, reduced the prothrombin time and increased endogenous thrombin potential. These agents may be viable options for reversal of therapeutic doses of rivaroxaban. SummaryBackground Oral activated factor X inhibitors such as rivaroxaban are widely used, but specific reversal agents are lacking. Although four-factor prothrombin complex concentrate (4F-PCC) and tranexamic acid (TXA) are sometimes used to manage serious bleeding, their efficacy is unknown. Prior studies in healthy subjects taking rivaroxaban revealed that 4F-PCC partially reverses the prolonged prothrombin time (PT), and fully restores the endogenous thrombin potential (ETP). The effect of TXA has not been evaluated. Methods In this double-blind, parallel-group study, 147 healthy volunteers given rivaroxaban 20 mg twice daily for 3 days were randomized after their morning dose on day 4 to receive intravenous 4F-PCC (50 IU kg−1), TXA (1.0 g), or saline. Standardized punch biopsies were performed at baseline and after 4F-PCC, TXA or saline administration. Reversal was assessed by measuring bleeding duration and bleeding volume at biopsy sites, and by determining the PT and ETP. Results As compared with saline, 4F-PCC partially reversed the PT and completely reversed the ETP, whereas TXA had no effect. Neither 4F-PCC nor TXA reduced bleeding duration or volume. All treatments were well tolerated, with no recorded adverse events. Conclusions Although 4F-PCC reduced the PT and increased the ETP in volunteers given supratherapeutic doses of rivaroxaban, neither 4F-PCC nor TXA influenced punch biopsy bleeding.

The rate of recurrent venous thromboembolism (VTE) in patients with a first unprovoked VTE who had a negative qualitative D-dimer test one month after stopping anticoagulant therapy was higher than expected in the D-dimer Optimal Duration Study (DODS).To determine whether quantitative D-dimer levels using a low threshold, age- and sex-specific thresholds, or repeated measurements, would improve identification of patients at low risk of recurrent VTE.D-dimer levels were quantified in banked samples from 307 patients in DODS who had a negative qualitative D-dimer test while on, and 1month after stopping, anticoagulant therapy and the rates of recurrent VTE were determined in patients with D-dimer levels below various predefined thresholds.The rate (per patient year) of recurrent VTE was: 5.9% with D-dimer levels250μg/l at one month; 5.2% with D-dimer levels between 250 and 499μg/l at one month; 5.0% with D-dimer levels less than predefined age- and sex-specific thresholds at one month; and 6.3% when D-dimer levels were500μg/l at both one and 7months after stopping anticoagulant therapy. These rates are similar to the overall event rate of 6.3% in patients who stopped treatment.Among unprovoked VTE patients who had a negative qualitative D-dimer test during and after anticoagulant therapy, low D-dimer thresholds, age and sex-adjusted thresholds or repeated measurements, did not identify subgroups with a very low rate of recurrence.

Transcranial Doppler-detected high-intensity transient signals (HITS) during cardiopulmonary bypass (CPB) surgery have been associated with postoperative neurocognitive dysfunction, suggesting microemboli in the brain could be a contributing factor. HITS occur despite administration of unfractionated heparin (UFH). This study was done to determine whether antithrombin-heparin covalent complex (ATH), a more potent anticoagulant than heparin, can reduce HITS during CPB. In a pig CPB model, ATH, UFH, or UFH + antithrombin (AT) was intravenously administered to female Yorkshire pigs after sternotomy. Twenty minutes later, hypothermic CPB was initiated and continued for 1.25 hours, then normothermia was re-established for 45 minutes. Protamine sulfate was given to neutralize the anticoagulants, and pigs were allowed to recover. HITS were monitored using an arterial flow probe placed over the carotid artery. Compared with UFH (300 or 1000 U/kg), ATH reduced the number of HITS during CPB in a dose-dependent manner. AT (3 mg/kg) + UFH (300 U/kg) resulted in an intermediate HITS rate between UFH and ATH (2 mg/kg in terms of AT). Examination of brain sections for emboli formation confirmed that, similar to HITS, number of thrombi decreased in direct proportion to ATH dosage. These results support the hypotheses that the majority of HITS represent thromboemboli and that ATH reduces emboli formation during CPB.

BACKGROUND In clinical practice, physicians are given the choice of selecting one of two dabigatran doses based on patient characteristics, with the lower dose typically used in patients at a higher risk of bleeding. OBJECTIVES The objectives of the study were to (i) estimate the inter- and intra-patient variability in dabigatran levels with 110 mg (DE110) and 150 mg (DE150) doses, (ii) examine the effect of physicians' dose selection on levels in DE110 and DE150 subgroups, and (iii) explore whether a single trough measurement identifies patients with extreme levels on subsequent visits. METHODS In this prospective observational study of 100 patients with atrial fibrillation (AF), peak and trough levels of dabigatran were measured with the Hemoclot(®) assay at baseline and every 2 months thereafter (maximum four visits). RESULTS Inter-patient variability in dabigatran levels (geometric coefficient of variation [gCV], 51-64%) was greater than intra-patient variability (gCV, 32-40%). Similar medians and distributions of levels were observed in DE110 and DE150 subgroups. Patients receiving DE110 were older, had lower renal function and weighed less than those receiving DE150. Up to 40% of patients whose trough levels were in the upper extremes, and up to 80% of patients whose trough levels were in the lower extremes at baseline, showed subsequent levels that fell in the middle quartiles. CONCLUSIONS Our data support the practice of selecting the dabigatran dose based upon clinical characteristics because it results in similar levels of drug exposure in patients given DE110 or DE150. They do not support the concept that a single Hemoclot(®) measurement reliably identifies patients with consistently high or low values.

The limitations of heparin and warfarin have prompted the development of new anticoagulant drugs for prevention and treatment of venous and arterial thromboembolism. Novel parenteral agents include synthetic analogs of the pentasaccharide sequence of heparin that mediates its interaction with antithrombin. Fondaparinux, the first synthetic pentasaccharide, is licensed for prevention of venous thromboembolism (VTE) after major orthopedic surgery and for initial treatment of patients with VTE. Idraparinux, a long-acting pentasaccharide that is administered subcutaneously once-weekly, is being compared with warfarin for treatment of VTE and for prevention of cardioembolic events in patients with atrial fibrillation. New oral anticoagulants include direct inhibitors of thrombin, factor Xa and factor IXa. Designed to provide more streamlined anticoagulation than warfarin, these agents can be given without routine coagulation monitoring. Ximelagatran, the first oral direct thrombin inhibitor, is as effective and safe as warfarin for prevention of cardioembolic events in patients with atrial fibrillation. However, ximelagatran produces a three-fold elevation in alanine transaminase levels in 7.9% of patients treated for more than a month, the long-term significance of which is uncertain. Whether other direct thrombin inhibitors or inhibitors of factors Xa or IXa also have this problem is under investigation. After a brief review of coagulation pathways, this paper focuses on new anticoagulants in advanced stages of clinical testing.

Summary. Background: The risk of recurrence is lower after treatment of an episode of venous thromboembolism associated with a transient risk factor, such as recent surgery, than after an episode associated with a permanent, or no, risk factor. Retrospective analyses suggest that 1 month of anticoagulation is adequate for patients whose venous thromboembolic event was provoked by a transient risk factor. Methods: In this double-blind study, patients who had completed 1 month of anticoagulant therapy for a first episode of venous thromboembolism provoked by a transient risk factor were randomly assigned to continue warfarin or to placebo for an additional 2 months. Our goal was to determine if the duration of treatment could be reduced without increasing the rate of recurrent venous thromboembolism during 11 months of follow-up. Results: Of 84 patients assigned to placebo, five (6.0%) had recurrent venous thromboembolism, compared with three of 81 (3.7%) assigned to warfarin, resulting in an absolute risk difference of 2.3%[95% confidence interval (CI) − 5.2, 10.0]. The incidence of recurrent venous thromboembolism after discontinuation of warfarin was 6.8% per patient-year in those who received warfarin for 1 month and 3.2% per patient-year in those who received warfarin for 3 months (rate difference of 3.6% per patient-year; 95% CI − 3.8, 11.0). There were no major bleeds in either group. Conclusion: Duration of anticoagulant therapy for venous thromboembolism provoked by a transient risk factor should not be reduced from 3 months to 1 month as this is likely to increase recurrent venous thromboembolism without achieving a clinically important decrease in bleeding.

See also Bompiani KM, Monroe DM, Church FC, Sullenger BA. A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity. This issue, pp 870–80.

Background The CURRENT-OASIS-7 and PLATO trials suggest that the benefit of clopidogrel is influenced by the dose of aspirin. Objective To explore a potential pharmacokinetic interaction between aspirin and clopidogrel, and determinants of clopidogrel active metabolite (AM) levels. Methods In part 1, using a 2 × 2 factorial design, we randomized patients to clopidogrel 600 mg loading dose (LD) followed by 150 mg day(-1) for 6 days and 75 mg day(-1) thereafter, or clopidogrel 300 mg LD followed by 75 mg day(-1) thereafter, and compared aspirin at 325 mg or 81 mg day(-1) . In part 2, patients were given a 600-mg clopidogrel LD, and were randomly allocated to aspirin 325 mg or 81 mg day(-1) . We combine the data from the two parts. Blood samples were collected 1 h after administration of the study drug. Results We randomized 302 patients (mean age 60.4 ± 9.9 years). Clopidogrel AM levels were similar in patients randomized to aspirin 325 or 81 mg (geometric mean, 12.70 ng mL(-1) ; 95% CI, 10.96-14.72 ng mL(-1) ; and geometric mean, 12.55 ng mL(-1) ; 95% CI, 10.80-14.58 ng mL(-1) ; P = 0.91). Blood levels of clopidogrel were lower in CYP2C19*2 loss-of-function (LOF) carriers compared with non-carriers (10.72 ng mL(-1) ; 95% CI, 8.83-13.01 ng mL(-1) ; and 15.21 ng mL(-1) ; 95% CI, 13.30-17.40 ng mL(-1) , respectively; P = 0.003) whereas levels in gain of function carriers and non-carriers were similar (13.31 ng mL(-1) ; 95% CI, 11.53-15.35 ng mL(-1) ; and 14.07 ng mL(-1) ; 95% CI, 11.74-16.87 ng mL(-1) , respectively; P = 0.4). Independent baseline predictors of clopidogrel AM levels were LOF genotype, body mass index, diabetes, proton pump inhibitor use and creatinine clearance, but accounted for only 20% of the variability in levels. Conclusion Aspirin dose does not predict clopidogrel AM levels 1 h post-LD. Most of the variability in clopidogrel AM levels is not explained by patient characteristics or CYP2C19 metabolizer status.

Background: Clinical situations occur where expedient assessment of the anticoagulant activity of the direct factor Xa (FXa) inhibitors is required. Although quantitative anti-FXa (FXa) assays can be used to measure plasma levels of apixaban or rivaroxaban, turnaround is often slow and many laboratories do not perform these assays. Objective: We compared the in vitro effects of apixaban and rivaroxaban on two readily available laboratory tests, the prothrombin time (PT) and activated partial thromboplastin time (APTT), performed with different reagents. We aimed to identify the most sensitive reagents. Methods: Rivaroxaban or apixaban was added to human plasma at a range of concentrations covering expected peak and trough levels, and concentrations were confirmed using calibrated anti-FXa assays. Samples were assayed with six PT and seven APTT reagents using different coagulometers. Results and Conclusions: TriniCLOT PT Excel S was the only reagent to demonstrate sensitivity to apixaban. All of the PT reagents were sensitive to rivaroxaban with TriniCLOT PT Excel S and HemosIL HS PLUS being the most sensitive. Sensitivity to rivaroxaban varied among APTT reagents; four reagents exhibited the greatest responsiveness, and of these, Actin FSL was the most responsive. Commonly used coagulation tests may be useful for assessing the anticoagulant effect of rivaroxaban but not of apixaban. The reason for the different effects of apixaban and rivaroxaban on the PT and APTT is unknown. Refereed/Peer-reviewed

Summary Background Four-factor prothrombin complex concentrates (PCCs), which contain factor II, FVII, FIX, and FX, have shown the potential to reverse the anticoagulant effect of rivaroxaban in healthy volunteers. The purpose of this study was to determine whether a three-factor PCC, which contains little FVII, has a similar effect. Methods and results We performed an open-label, single-center, parallel-group study comparing the effect of a three-factor PCC (Profilnine SD) with that of a four-factor PCC (Beriplex P/N) on the pharmacodynamics of rivaroxaban in 35 healthy volunteers. After receiving 4 days of rivaroxaban 20 mg twice daily to obtain supratherapeutic steady-state concentrations, volunteers were randomized to receive a single 50 IU kg−1 bolus dose of four-factor PCC, three-factor PCC or saline 4 h after the morning dose of rivaroxaban on day 5, and the effects of these interventions on prothrombin time and thrombin generation were determined. Within 30 min, four-factor PCC reduced mean prothrombin time by 2.5–3.5 s, whereas three-factor PCC produced only a 0.6–1.0-s reduction. In contrast, three-factor PCC reversed rivaroxaban-induced changes in thrombin generation more than four-factor PCC. Conclusions This study demonstrates the potential of both three-factor and four-factor PCCs to at least partially reverse the anticoagulant effects of rivaroxaban in healthy adults. The discrepant effects of the PCC preparations may reflect differences in the procoagulant components present in each.

Alterations in the cellular redox potential by homocysteine promote endothelial cell (EC) dysfunction, an early event in the progression of atherothrombotic disease. In this study, we demonstrate that homocysteine causes endoplasmic reticulum (ER) stress and growth arrest in human umbilical vein endothelial cells (HUVEC). To determine if these effects reflect specific changes in gene expression, cDNA microarrays were screened using radiolabeled cDNA probes generated from mRNA derived from HUVEC, cultured in the absence or presence of homocysteine. Good correlation was observed between expression profiles determined by this method and by Northern blotting. Consistent with its adverse effects on the ER, homocysteine alters the expression of genes sensitive to ER stress (ie, GADD45, GADD153, ATF-4, YY1). Several other genes observed to be differentially expressed by homocysteine are known to mediate cell growth and differentiation (ie, GADD45, GADD153, Id-1, cyclin D1, FRA-2), a finding that supports the observation that homocysteine causes a dose-dependent decrease in DNA synthesis in HUVEC. Additional gene profiles also show that homocysteine decreases cellular antioxidant potential (glutathione peroxidase, NKEF-B PAG, superoxide dismutase, clusterin), which could potentially enhance the cytotoxic effects of agents or conditions known to cause oxidative damage. These results successfully demonstrate the use of cDNA microarrays in identifying homocysteine-respondent genes and indicate that homocysteine-induced ER stress and growth arrest reflect specific changes in gene expression in human vascular EC.

Heparin has been used extensively for prophylaxis and treatment of deep vein thrombosis. However, heparin has several limitations including a short intravenous half-life, inability to inhibit clot-bound thrombin, and bleeding. We have developed a covalent antithrombin-heparin complex (ATH) that has a longer intravenous half-life and greater anticoagulant activity than heparin. The antithrombotic activity of ATH was tested in a rabbit jugular vein thrombosis treatment model. Administration of ATH caused a 17% reduction in clot weight compared with an increase of 24, 60, 172 and 135% for administration of antithrombin plus heparin, heparin, antithrombin and saline, respectively. Clot weight and fibrin accretion were both significantly lower in the ATH group than in the antithrombin plus heparin group (P < 0.05). The peak anti-factor-Xa activity was fourfold higher in the ATH group than in the antithrombin plus heparin group. Using a rabbit bleeding ear model, there was no significant difference in cumulative blood loss between ATH and antithrombin plus heparin groups, at similar plasma anti-factor-Xa levels. In conclusion, ATH has superior antithrombotic activity and similar bleeding effect compared with heparin on a mass basis. The enhanced antithrombotic activity of ATH may be a result of its increased anticoagulant activity or its longer half life, or both.

Wells PS, Hirsh J, Anderson DR, Lensing AWA, Foster G, Kearon C, Weitz J, D'Ovidio R, Cogo A, Prandoni P, Girolami A, Ginsberg JS (Ottawa Civic Hospital, Ottawa, Hamilton Civic Hospitals Research Centre, Henderson General Hospital, and McMaster University, Hamilton, Ontario, and Victoria General Hospital, Nova Scotia, Canada; Instituto di Semeioti Medica, Padua, Italy; and Academic Medical Centre, Amsterdam, the Netherlands). A simple clinical model for the diagnosis of deep-vein thrombosis combined with impedance plethysmography: potential for an improvement in the diagnostic process. J Intern Med 1998; 243: 15–23. Objectives We recently demonstrated the utility of a clinical model combined with ultrasonography to assist the diagnostic approach in patients with suspected deep-vein thrombosis (DVT). In this study we also sought to demonstrate that the model is useful with impedance plethysmography, a less accurate and less utilized diagnostic test. The original clinical model is slightly cumbersome to use; thus at the completion of the study we attempted to develop a simpler scoring system with a goal of maintaining accuracy. Design An open, nonrandomized, multicentre trial. Setting Three centres, two in Canada, and one in Italy. Subjects Ambulatory patients with suspected deep-vein thrombosis. Interventions All patients were assessed clinically to determine the probability for deep-vein thrombosis prior to performing impedance plethysmography and venography. We compared the accuracy of impedance plethysmography between the three pretest probability categories of high, moderate and low. All of the above were performed and interpreted by independent observers. When the study was completed, we revised the clinical model by first performing a simple regression analysis then a multiple logistic regression analysis; a scoring system was devised using the latter. Results Impedance plethysmography is significantly more sensitive and less specific for all DVT in patients with high pretest probability for deep-vein thrombosis (P= 0.001). The post- test probability (positive predictive value) for deep-vein thrombosis with an abnormal impedance plethysmography result was significantly different (P= 0.0001) between the three pretest probability categories. Multiple regression analysis has provided a new model with only nine variables and a simple scoring system. The retrospective application of the revised clinical model, which is simpler to use, suggests it will provide similar results as the original clinical model when combined with impedance plethysmography. The combination of impedance plethysmography and the clinical model suggests patients are likely to have false positive results if they have a low or moderate pretest probability for deep-vein thrombosis and false negative results if the pretest probability is high. The combination of a low pretest probability and a normal impedance plethysmography result may exclude the need for serial testing, and represented more than 50% of our patient population. Conclusions The use of the clinical model in conjunction with impedance plethysmography would decrease the number of false positive and negative diagnoses and could markedly decrease the need for serial impedance plethysmography. Combining the clinical model with impedance plethysmography could overcome the fact that impedance plethysmography is clearly less accurate than venous ultrasound imaging. The use of the revised clinical model may increase acceptability and utility, but prospective testing is required before widespread use.

Background Low-molecular-weight heparin is known to be safe and effective for the initial Treatment of patients with proximal deep-vein thrombosis. However, its application to patients with pulmonary embolism or previous episodes of thromboembolism has not been studied.Methods We randomly assigned 1021 patients with symptomatic venous thromboembolism to fixed-dose, subcutaneous low-molecular-weight heparin (reviparin sodium) or adjusted-dose, intravenous unfractionated heparin. Oral anticoagulant therapy with a coumarin derivative was started concomitantly and continued for 12 weeks, Approximately one third of the patients had associated pulmonary embolism, The outcome events studied over the 12 weeks were symptomatic recurrent venous thromboembolism, major bleeding, and death. We sought to determine whether low-molecular-weight heparin is at least equivalent to unfractionated heparin in patients with venous thromboembolism.Results Twenty-seven of the 510 patients assigned to low-molecular-weight heparin (5.3 percent) had recurrent thromboembolic events, as compared with 25 of the 511 patients assigned to unfractionated heparin (4.9 percent). The difference of 0.4 percentage point indicates that the two therapies have equivalent value according to our predetermined definition of equivalence. Sixteen patients assigned to low-molecular-weight heparin (3.1 percent) and 12 patients assigned to unfractionated heparin (2.3 percent) had episodes of major bleeding (P=0.63), and the mortality rates in the two groups were 7.1 percent and 7.6 percent, respectively (P=0.89).Conclusions Fixed-dose, subcutaneous low-molecular-weight heparin is as effective and safe as adjusted-dose, intravenous unfractionated heparin for the initial management of venous thromboembolism, regardless of whether the patient has pulmonary embolism or a history of venous thromboembolism. (C) 1997, Massachusetts Medical Society.

Summary. Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half-life. Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet-mediated events during thrombus formation. Methods: The affinities of Diannexin and annexin A5 for PS-containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet-fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laserinduced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results: Diannexin and annexin A5 bind PS with KD values of 0.6 and 5 nM, respectively, and both bind to the same subpopulation of PS-exposing platelets. Diannexin inhibited thrombin generation and platelet-fibrin clot formation in vitro at 10 nM (P < 0.05–0.001 compared with control), and reduced platelet accumulation at 1 l gg )1 (P < 0.05) and activation at 0.25 l gg )1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 l gg )1 (P

SummaryIn order to determine if there is a relationship between antiphospholipid antibodies and reduced free protein S levels, we evaluated 21 patients who had an antiphospholipid antibody but had neither a history of venous thromboembolism nor systemic lupus erythematosus (cases) and 55 matched controls, who did not have an antiphospholipid antibody, a history of thrombosis or systemic lupus erythematosus. Cases and controls had similar protein C and antithrombin levels. Six of 21 cases had reduced free protein S antigen levels, compared to 5 of 55 controls (x 2 = 5.823 p

Little is known with regard to efficacy of heparin as an adjunct to fibrinolytics under conditions of severe vascular damage. In this study, we compared the effects of unfractionated heparin (UH), low-molecular weight heparin (LMWH), and recombinant desulfohirudin (HIR) in combination with streptokinase (SK) in such settings.We used an established rabbit model, in which thrombosis, critical stenosis, and vascular wall damage were introduced to a segment of the abdominal aorta and the effects of the respective therapies were assessed by time to patency (TTP in minutes), cumulative patency (CP (%)), lysis of original clot (CL (%)), and net clot accretion (NCA (%)). Treatments were administered over 90 min at the following doses: SK: 33,000 U/kg, UH: 125-250 U/kg, LMWH: 1.25-2.5 mg/kg, HIR: 0.25-0.55 mg/kg.Unexpectedly, UH and LMWH had a paradoxical and detrimental effect on SK-mediated recanalization as measured by both TTP and CP. Thus, administration of SK vs. SK+UH or SK+LMWH resulted in TTP values of 43+/-8 min vs. 70+/-11 min (p0.05) and 67+/-12 min. (p0.08), respectively. For CP, the corresponding values were 21+/-7%, 0.5+/-0.3% and 9+/-8%. This delay in vessel recanalization occurred despite excessive systemic anticoagulation (activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) ratios6 and34, respectively). Of interest, both heparinoids completely inhibited SK-induced fibrinogen consumption (FC). In contrast, recombinant desulfohirudin (HIR) shortened SK-induced TTP (4.97+/-0.81 min) without preserving fibrinogen.Our findings suggest that caution needs to be exercised, when using the combination of SK and heparinoids for the treatment of arterial thrombosis under conditions of severe vascular damage and stenosis.

BACKGROUND: Using data from AMPLIFY, a phase III trial comparing apixaban with conventional anticoagulant treatment in patients with venous thromboembolism (VTE), we performed subgroup analyses to compare the efficacy and safety of these regimens in patients with and without active cancer at baseline. Active cancer was defined as cancer that was diagnosed or treated within the past 6 months. METHODS: Patients with symptomatic VTE were randomized to a 6-month course of apixaban (10mg BID for 7 days followed by 5mg BID) or conventional treatment consisting of enoxaparin (1mg/kg BID for at least 5 days) followed by doseadjusted warfarin (target INR, 2-3). Cancer patients for whom long-term low-molecular-weight heparin (LMWH) was planned were excluded. The primary efficacy outcome was symptomatic VTE or VTE-related death. The intent-to-treat efficacy analysis included all randomized subjects with a nonmissing primary endpoint. The primary safety outcome was ISTH-defined major bleeding up to 2 days after stopping study drug in all randomized subjects who received at least one dose of study medication. All outcomes were adjudicated by an independent committee blinded to treatment assignment. RESULTS: Of 5395 patients randomized, 169 (3.1%) had active cancer. Baseline characteristics in these patients were similar between the two treatment groups. The median duration of treatment was 167 and 168 days in the apixaban and warfarin groups, respectively. In patients with active cancer at entry, recurrent VTE occurred in 3 of 81 (3.7%) patients in the apixaban group and in 5 of 78 (6.4%) in the warfarin group (relative risk [RR], 0.56; 95% CI, 0.132.37); major bleeding occurred in 2 of 87 (2.3%) and 4 of 80 (5.0%) patients, respectively (RR, 0.45; 95% CI, 0.082.46). In patients without cancer at entry, recurrent VTE occurred in 56 of 2528 (2.2%) and in 66 of 2557 (2.6%) patients in the apixaban and warfarin group, respectively (RR, 0.86; 95% CI, 0.60-1.22), whereas major bleeding occurred in 13 of 2589 (0.5%) and in 45 of 2609 (1.7%) patients in the apixaban and warfarin group, respectively (RR, 0.29; 95% CI, 0.16-0.54). CONCLUSION: Although the number of cancer patients was small, the results in this pre-specified subgroup are consistent with the overall findings and suggest that apixaban is as effective as conventional therapy in VTE patients with active cancer, and is associated with less bleeding. Additional studies are needed to compare the efficacy and safety of apixaban and LMWH for VTE treatment in cancer patients. Pfizer, BMS

Arterial and venous thrombosis are major causes of morbidity and mortality. Arterial thrombosis is the most common cause of myocardial infarction, stroke, and limb gangrene. Venous thrombosis leads to potentially fatal pulmonary embolism and to post-phlebitic syndrome. Because arterial thrombi consist of platelet aggregates held together by small amounts of fibrin, strategies to inhibit arterial thrombogenesis focus mainly on blocking platelet function, but often include anticoagulants to prevent fibrin deposition. Anticoagulants are used for the prevention and treatment of venous thrombosis because venous thrombi consist mainly of fibrin and red blood cells. This paper (1) reviews arterial and venous thrombogenesis, (2) outlines new anticoagulant strategies, and (3) provides clinical perspectives on these new strategies.

To determine whether fibrinolytic testing predicts recurrent venous thrombosis, we have performed a prospective cohort study in which 303 patients with a first episode of venous thromboembolism underwent comprehensive fibrinolytic testing while receiving oral anticoagulants, and after anticoagulants had been discontinued. They were then followed for up to 3 years for recurrent venous thrombosis. No systematic differences in the levels or activity of type 1 plasminogen activator inhibitor (PAI-1), tissue plasminogen activator (tPA) or euglobulin clot lysis times were detected between patients who did, or did not, suffer recurrent thrombosis. There were also no differences in these variables when patients whose initial thrombosis was idiopathic were compared to patients whose thrombosis occurred in the setting of a known thrombotic risk factor. Based on these results, neither measuring fibrinolytic parameters in patients with venous thromboembolism, nor modification of treatment based on the results of such testing, are justified. Our study also confirms that patients with idiopathic venous thromboembolism have a high risk of recurrence.

Plasmin generation is localized to the fibrin surface because tissue-type plasminogen activator (t-PA) and plasminogen bind to fibrin, an interaction that stimulates plasminogen activation over a hundred-fold. To ensure efficient fibrinolysis, plasmin bound to fibrin is protected from inhibition by alpha2-antiplasmin. (DD)E, a major soluble degradation product of cross-linked fibrin that is a potent stimulator of t-PA, compromises the fibrin-specificity of t-PA by promoting systemic activation of plasminogen. In this study we investigated whether (DD)E also protects plasmin from inhibition by alpha2-antiplasmin, facilitating degradation of this soluble t-PA effector. (DD)E and fibrin reduce the rate of plasmin inhibition by alpha2-antiplasmin by 5- and 10-fold, respectively. Kringle-dependent binding of plasmin to (DD)E and fibrin, with Kd values of 52 and 410 nM, respectively, contributes to the protective effect. When (DD)E is extensively degraded by plasmin, yielding uncomplexed fragment E and (DD), protection of plasmin from inhibition by alpha2-antiplasmin is attenuated. These studies indicate that (DD)E-bound plasmin, whose generation reflects the ability of (DD)E to stimulate plasminogen activation by t-PA, has the capacity to degrade (DD)E by virtue of its resistance to inhibition. This provides a mechanism to limit the concentration of (DD)E and maintain the fibrin-specificity of t-PA.

Although heparin is widely used to treat arterial thrombosis, it has limitations in this setting. These limitations reflect heparin's inability to inactivate fibrin-bound thrombin, a major stimulus for thrombus growth, and the fact that heparin is neutralized by platelet factor 4, large quantities of which are released from platelets at the site of plaque rupture. Heparin also has a propensity to bind non-specifically to other plasma proteins. Because plasma levels of these heparin-binding proteins vary from patient to patient, the anticoagulant response to heparin is unpredictable and careful laboratory monitoring is necessary to ensure that an adequate anticoagulant effect is achieved. Direct thrombin inhibitors, such as bivalirudin and hirudin, overcome many of the limitations of heparin. These agents inhibit fibrin-bound thrombin, as well as fluid-phase thrombin. Direct thrombin inhibitors also produce a more predictable anticoagulant response than heparin because they do not bind to plasma proteins and are not neutralized by platelet factor 4. Bivalirudin appears to have a wider therapeutic window than hirudin. Because this may permit administration of higher doses of bivalirudin, this agent may also have an efficacy advantage over hirudin. Differences observed between hirudin and bivalirudin demonstrate that not all direct thrombin inhibitors have the same risk-benefit profile.

Women with a history of venous thromboembolism may be at increased risk for venous thromboembolic events during pregnancy. In these women, the decision to give or withhold heparin in the antepartum period is controversial, because accurate estimates of the frequency of recurrent thromboembolic events if antepartum heparin is withheld are not available.We prospectively studied 125 pregnant women with a single previous episode of venous thromboembolism. Antepartum heparin was withheld, but anticoagulant therapy was given for four to six weeks post partum. Our primary objective was to determine the rate of antepartum recurrence of venous thromboembolism. Laboratory studies were performed to identify thrombophilia in 95 women.Three of the 125 women (2.4 percent) had an antepartum recurrence of venous thromboembolism (95 percent confidence interval, 0.2 to 6.9 percent). There were no recurrences in the 44 women who had no evidence of thrombophilia and who also had a previous episode of thrombosis that was associated with a temporary risk factor. Among the 51 women with abnormal laboratory results or a previous episode of idiopathic thrombosis, or both, 3 (5.9 percent) had an antepartum recurrence of venous thromboembolism (95 percent confidence interval, 1.2 to 16.2 percent).The risk of recurrent antepartum venous thromboembolism in women with a history of venous thromboembolism is low, and therefore routine antepartum prophylaxis with heparin is not warranted.

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.

To compare effects of heparin and hirudin on biochemical markers of coagulation.Patients (n=395) with unstable angina or myocardial infarction without ST elevation were randomized to a 72-h infusion of one of three regimens: unfractionated heparin (bolus of 5000 IU followed by an infusion of 1200 IU. h(-1)), low-dose hirudin (HBW 023; 0.2 mg. kg(-1)bolus followed by 0.10 mg. kg(-1). h(-1)) or medium-dose hirudin (0.4 mg. kg(-1)bolus followed by 0.15 mg. kg(-1). h(-1)). Infusions were adjusted to maintain an activated partial thromboplastin time of between 60-100 s. Activated partial thromboplastin time, prothrombin fragment 1.2 (F1.2), thrombin antithrombin III complex and D-dimer were measured before, during and after the infusion. Median activated partial thromboplastin time was similar in the two groups early on, but was significantly lower in the heparin group than in the combined hirudin group 48 h after starting the infusion (53 s and 75 s, respectively;P0.001), and 6 h after stopping (31 s and 46 s, respectively;P0.001). Median F1.2 levels were not significantly different between the groups during the infusion. Median thrombin antithrombin III levels in the heparin and hirudin groups were 2.8 microg. l(-1)and 2.3 microg. l(-1), respectively, at 6 h (P0.001), and 3.0 microg. l(-1)and 2.3 microg. l(-1), respectively, at 48 h (P0.001). Median D-dimer levels were 320 ng. ml(-1)and 260 ng. ml(-1)48 h after starting the infusion in the heparin and hirudin groups, respectively (P0.001), and 415 ng. ml(-1)and 280 ng. ml(-1), respectively (P0.001) 6 h after stopping. D-dimer levels were significantly elevated above baseline values in both groups 24-48 h after stopping the infusions.The greater reduction of thrombin antithrombin III and D-dimer during the hirudin infusion supports the hypothesis that hirudin is a more potent antithrombin agent than heparin. Increased D-dimer levels after stopping heparin or hirudin suggest that there is an ongoing pro-coagulant state. These results point to the greater efficacy of hirudin in preventing early clinical events (death, myocardial infarction and refractory ischaemia) compared with heparin that have been observed in large randomized trials. Persistent activation of coagulation afterstopping infusions in our study suggests that a longer course of antithrombotic treatment may be needed to pacify the thrombus.

Most of the major advances in thrombosis research have occurred in the last 50 years, reflecting progress in biomedical sciences and clinical trials methodology. Improved understanding of the mechanisms of thrombogenesis has led to the discovery of a plethora of new antithrombotic agents that target many of the key steps in blood coagulation and platelet activation. Although most of these compounds are still under development, low-molecular-weight heparins (LMWH), glycoprotein (GP) IIb/IIIa receptor antagonists, and inhibitors of the adenosine diphosphate (ADP) receptor on platelets have already established their niche in the clinic. The vessel wall has emerged as a major player, both in protecting against and in promoting thrombosis, and as we approach the new millennium, compounds are being developed that have the potential to prevent and treat thrombosis by modulating vessel wall function.

Alterations in the cellular redox potential by homocysteine promote endothelial cell (EC) dysfunction, an early event in the progression of atherothrombotic disease. In this study, we demonstrate that homocysteine causes endoplasmic reticulum (ER) stress and growth arrest in human umbilical vein endothelial cells (HUVEC). To determine if these effects reflect specific changes in gene expression, cDNA microarrays were screened using radiolabeled cDNA probes generated from mRNA derived from HUVEC, cultured in the absence or presence of homocysteine. Good correlation was observed between expression profiles determined by this method and by Northern blotting. Consistent with its adverse effects on the ER, homocysteine alters the expression of genes sensitive to ER stress (ie, GADD45, GADD153, ATF-4, YY1). Several other genes observed to be differentially expressed by homocysteine are known to mediate cell growth and differentiation (ie, GADD45, GADD153, Id-1, cyclin D1, FRA-2), a finding that supports the observation that homocysteine causes a dose-dependent decrease in DNA synthesis in HUVEC. Additional gene profiles also show that homocysteine decreases cellular antioxidant potential (glutathione peroxidase, NKEF-B PAG, superoxide dismutase, clusterin), which could potentially enhance the cytotoxic effects of agents or conditions known to cause oxidative damage. These results successfully demonstrate the use of cDNA microarrays in identifying homocysteine-respondent genes and indicate that homocysteine-induced ER stress and growth arrest reflect specific changes in gene expression in human vascular EC.

Although it is well established that long-term heparin therapy causes osteoporosis, it is unknown whether heparin-induced bone loss is reversible when heparin treatment is stopped. To address this question, we randomized rats to once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g) or saline for 28 days and then followed the rats for an additional 28 days off treatment. Based on histomorphometric analysis of the distal third of the right femur proximal to the epiphyseal growth plate, 1.0 U/g heparin caused a 30% loss in cancellous bone volume over the first 28 days. This was accompanied by a 137% increase in osteoclast surface and a 60% decrease in both osteoblast and osteoid surface. One month after cessation of heparin treatment, no recovery in these parameters was observed. Similarly, serum levels of alkaline phosphatase, a biochemical marker of bone formation, which continued to decrease over the course of heparin treatment, showed no signs of recovery in the subsequent 28 days off treatment. To explore the mechanism responsible for the prolonged effect of heparin on bone, we repeated the experiment giving 125I-labeled heparin in place of unlabeled heparin. 125I-labeled heparin was found to accumulate in bone during the course of its administration, and be retained in bone for at least 56 days after stopping heparin treatment. These findings suggest that heparin-induced osteoporosis is not rapidly reversible because heparin is sequestered in bone for an extended period.