Platelet activation in patients with peripheral vascular disease: reproducibility and comparability of platelet markers.

BACKGROUND: Many markers of platelet activation have been described but their reproducibility and comparability in patient populations are poorly defined. OBJECTIVES: We sought to compare markers of platelet and monocyte activation with platelet-monocyte aggregates, a proposed gold standard of in vivo platelet activation, and assess their reproducibility in patients with peripheral arterial disease: a population with substantial platelet activation, inflammation and risk of thrombotic events. PATIENTS/METHODS: Thirty patients with peripheral vascular disease attended on two occasions to permit within-day and between-day comparisons. In vivo platelet and monocyte activation were determined by flow-cytometric quantification of platelet-monocyte aggregation, platelet surface expression of P-selectin and CD40L, platelet-derived microparticles, and monocyte surface expression of CD40 and CD11b. Plasma concentrations of platelet-derived microparticles, soluble P-selectin and CD40L were measured by enzyme-linked immunosorbant assays. RESULTS: Platelet-monocyte aggregation (36.7±7.86%), and platelet surface expression of P-selectin (5.8±1.65%) and CD40L (3.3±1.45%) demonstrated comparable within-day (mean difference±co-efficient of reproducibility; 0.9±15.4%, 0.21±1.65% and 0.2±2.8% respectively) and between-day reproducibility (2.0±12.4%, 0.10±2.25% and 0.9±6.4% respectively). Platelet-monocyte aggregates correlated well with other platelet (r=0.30-0.50, P<0.02) and monocyte (r=0.27-0.47, P<0.03) activation markers. Flow cytometric and assay quantified platelet-derived microparticles showed poorer reproducibility (co-efficient of reproducibility >40). CONCLUSIONS: In patients with peripheral arterial disease, measurements of platelet-monocyte aggregates have good reproducibility and consistently reflect other markers of platelet and monocyte activation.

Syndecan-1 plasma levels during coronary artery bypass surgery with and without cardiopulmonary bypass.

The glycocalyx covering the endothelium is shed during ischemia and reperfusion. The shedding is accompanied by increased levels of the glycocalyx component syndecan-1 in the circulation. Our aim was to compare plasma levels of syndecan-1 in patients undergoing coronary artery bypass grafting (CABG), with or without the use of cardiopulmonary bypass (CPB). Syndecan-1 plasma concentrations were measured in patients undergoing CABG on-pump (nA =A 22) or off-pump (nA =A 22). The syndecan-1 concentration increased significantly from 29.5 +/- 4.6 ng/mL at baseline to 98.7 +/- 9.8 ng/mL (pA

New biomarkers in an acute model of live Escherichia coli-induced sepsis in pigs.

We developed a live Escherichia coli model of acute sepsis in pigs with emphasize on biomarkers reflecting the early inflammatory response of sepsis. Healthy pigs, 25-35 kg, were challenged intravenously (IV) (n = 12) or intrapulmonary (n = 6) with live E. coli and observed for 3 and 5 h respectively. Control pigs received culture medium (n = 6 + 3). Haemodynamic parameters and a broad panel of inflammatory mediators were measured. The dose of bacteria was carefully titrated to obtain a condition resembling the early phase of human septic shock. The IV group displayed a pro-inflammatory response [significant increase in tumour necrosis factor-alpha, interleukin (IL)-6 and IL-8] and an early anti-inflammatory response (significant increase in IL-10). For the first time, we demonstrate a significant increase in IL-12 and matrix metalloproteinase-9 (MMP) early in pig sepsis. Coagulation was activated (significant increase in thrombin-antithrombin complexes) and there was a significant decrease in the serum proteins suggesting capillary leakage. Haemodynamic parameters reflected a septic condition with significant decrease in systemic blood pressure, increases in heart rate, pulmonary artery pressure and base deficit. None of these changes was observed in the control group. Interleukin-1beta and vascular endothelial growth factor increased in both groups. Nitric oxide measurements suggested an initial pulmonary vascular endothelial inflammatory response. The intrapulmonary group, which did not resemble septic condition, showed a substantial increase in MMP-9. In this porcine model of sepsis, IL-12 and MMP-9 were detected for the first time. These biomarkers may have an impact in the understanding and future treatment of sepsis.

No case of COX-1-related aspirin resistance found in 289 patients with symptoms of stable CHD remitted for coronary angiography.

OBJECTIVE: To assess the prevalence of a lacking aspirin effect on cyclooxygenase-1 (COX-1) ("aspirin resistance") in patients with symptomatic, stable coronary heart disease (CHD) using test methods directly reflecting inhibition of COX-1. MATERIAL AND METHODS: Arachidonic acid (AA)-induced platelet aggregation and plasma thromboxane B2 (TXB2) were determined twice 3 weeks apart - prior to elective coronary angiography - in 289 patients on 75 or 160 mg aspirin daily, all prompted to take aspirin before testing. Subjects who demonstrated lacking any effect of aspirin (>/=20 % AA-induced aggregation) on one or both occasions were later given a third test. Forty-two patients not taking aspirin were used as TXB2 controls. RESULTS: Eleven (3.8 %) had aggregation > or = 20 % in at least one of the two initial tests, but only two on both occasions. During the third test, all 11 patients had aggregation <20 %. The TXB2 distributions in controls and study patients differed markedly (mean 173 versus 19 pg/mL). Taking 45 pg/mL as the TXB2 cut-off level, sensitivity and specificity for detecting subjects taking aspirin were 90 % and 89 %, respectively. The area under the ROC curve was 0.96. CONCLUSION: Repeated AA-induced platelet aggregometry showed that COX-1 could be blocked by low-dose aspirin in all 289 tested patients, suggesting that aspirin resistance is rare in patients with stable CHD.

Clopidogrel increases expression of chemokines in peripheral blood mononuclear cells in patients with coronary artery disease: results of a double-blind placebo-controlled study.

BACKGROUND: Chemokines and platelet activation are both important in atherogenesis. Platelet inhibitors are widely used in coronary artery disease (CAD), and we hypothesized that the platelet inhibitor clopidogrel could modify chemokines in CAD patients. OBJECTIVES: We sought to investigate the effect of clopidogrel on the expression of chemokines and chemokine receptors in peripheral blood mononuclear cells (PBMC) in CAD patients. PATIENTS/METHODS: Thirty-seven patients with stable angina were randomized to clopidogrel (n = 18) or placebo (n = 19). PBMC, blood platelets and plasma were collected at baseline and after 7-10 days in the patients, and in 10 healthy controls. mRNA levels of chemokines and chemokine receptors in PBMC were analyzed by ribonuclease protection assays and real-time reverse transcriptase polymerase chain reaction. Platelet activation was studied by flow cytometry. RESULTS: (i) At baseline, the gene expression of the regulated on activation normally T-cell expressed and secreted (RANTES) chemokines and macrophage inflammatory peptide (MIP)-1beta in PBMC, the expression of CD62P and CD63 on platelets and the levels of platelet-derived microparticles (PMP) were elevated in angina patients comparing healthy controls; (ii) markers of platelet activation were either reduced (CD63) or unchanged (CD62P, PMP, beta-thromboglobulin) during clopidogrel therapy; (iii) in contrast, clopidogrel significantly up-regulated the gene expression of RANTES and MIP-1beta in PBMC, while no changes were found in the placebo group; (iv) a stable adenosine 5'-diphosphate metabolite attenuated the release of MIP-1beta, but not of RANTES, from activated PBMC in vitro. CONCLUSIONS: Even if we do not argue against a beneficial role for clopidogrel in CAD, our findings may suggest potential inflammatory effects of clopidogrel in CAD.

Children with acute lymphoblastic leukaemia have high plasma levels of total homocysteine at time of diagnosis.

OBJECTIVE: Cancer can induce venous thromboembolic complications for various reasons. As part of a greater study, acquired and congenital prothrombotic risk factors were investigated in children with leukaemia or non-Hodgkin's lymphoma and compared with similar investigations in children with congenital heart defects. MATERIAL AND METHODS: Blood samples were taken from 60 children with newly diagnosed leukaemia or lymphoma and 133 children with congenital heart defects in the course of a scheduled cardiac catheterization. When children with cancer were in remission, analyses of acquired prothrombotic risk factors were repeated. Children with cancer were observed for symptoms of thromboembolism throughout their treatment period. RESULTS: Total homocysteine levels were significantly raised in children with cancer (median value 10.0 micromol/L) as compared with the levels in children with congenital heart diseases (5.0 micromol/L) (p<0.001), while children with acute lymphoblastic leukaemia had the highest values. The median level of lipoprotein(a) was slightly increased in children with newly diagnosed leukaemia or lymphoma (105 mg/L versus 100 mg/L, p<0.001), and levels of coagulation inhibitors were higher (p<0.001). Total homocysteine levels normalized when children attained remission of cancer disease. Two children had symptoms of acute thrombosis. CONCLUSIONS: Raised concentrations of total homocysteine were frequent in children with newly diagnosed cancer, but this normalized when the children were in remission. The clinical significance of our observations and the impact on venous thromboembolism have yet to be defined.

Acquired haemophilia: management of bleeds and immune therapy to eradicate autoantibodies.

Acquired haemophilia is a rare, but often severe bleeding disorder caused by autoantibodies against a coagulation factor, usually factor VIII (FVIII). Between 1997 and 2004 we observed 14 patients (mean age of 78 years) with acquired haemophilia. The aim of the present study was to investigate the effect of activated prothrombin complex concentrate (aPCC) for bleeds and the response to corticosteroids and cyclophosphamide to eradicate the offending autoantibodies. The most common clinical presentations were severe profuse bruising (12) and haematuria (5). Ten patients were classified as idiopathic. At the time of diagnosis all patients had a very low FVIII level, and one patient also showed factor IX < 1%. High levels of antibodies to FVIII varying from 10 to 1340 Bethesda units (BU) and prolonged activated partial thromboplastin time were disclosed in all patients. Eight severe bleeds were treated with aPCC (FEIBA) at a dosage of 70 IU kg(-1) every 8 h until haemostasis. Ten patients received corticosteroids and cyclophosphamide as immunomodulatory therapy. Effective haemostasis was achieved in all bleeds after aPCC. Ten of 11 patients responded either completely or partially to the immunomodulatory regime within 6 months. Five patients achieved complete response (CR) whereas partial responses were seen in five patients. The anti-CD20 monoclonal antibody rituximab was given to two patients in conventional doses and a CR was seen in one patient. aPCC is effective in treating acute bleeds in patients with acquired haemophilia with high inhibitor levels. The combination of oral corticosteroids and cyclophosphamide seems to be effective to eradicate the inhibitor.

Effects of platelets and platelet-derived material on the activated partial thromboplastin time (Cephotest) coagulation test.

OBJECTIVE: The Cephotest is an activated partial thromboplastin time (APTT) test used to measure the activity of the intrinsic pathway of coagulation. To perform this test, blood is usually centrifuged to obtain plasma that is almost without erythrocytes and leucocytes and with only a minimal amount of platelets. MATERIAL AND METHODS: In the present experiments blood was centrifuged at different speeds to produce either platelet-poor plasma (PPP) or platelet-rich plasma (PRP). PPP and PRP obtained from the same whole blood samples from each of several persons were tested in pairs using the standard Cephotest reagent to observe the consequences of Cephotest being performed on plasma containing platelets. The same procedure was used with a Cephotest reagent with a reduced concentration of phosphatidylserine. In succeeding experiments the PRP was preincubated with SFLLRN or Ca-ionophore to activate the platelets, a procedure also known to produce platelet-derived microparticles. PPP and PRP were compared by thrombin time and reptilase time tests to find out at which stage platelets might influence the Cephotest. RESULTS: The results showed that the platelets did influence Cephotest when using both the regular reagent and the phospholipid-reduced agent. When using the regular reagent, PRP showed a tendency towards a longer APTT than PPP. It is suggested that this was caused by platelets consuming some of the first traces of thrombin generated. CONCLUSIONS: When using the phospholipid-reduced reagent, PRP showed a shorter APTT than PPP, probably because the platelets contributed phosphatidylserine to the system. When the platelets were activated before testing, their effects on the tests were increased. Microparticles that formed during platelet activation may have contributed to these effects.

Clinical implications of red blood cell and platelet storage lesions: an overview.

Both red blood cells and platelets undergo lesions upon storage which affect their function and possibly their clinical outcome. Some of these lesions are reversible, others not. Improved additive solutions and leukocyte depletion can delay the appearance of storage lesions. In addition, cellular apoptosis leads to numerous mitochondrial and surface changes during storage which have the potential to induce immune suppression by tuning down the innate immune system. This overview highlights some laboratory and clinical aspects of red cell and platelet storage lesions.

Activated prothrombin complex concentrate (FEIBA) treatment during surgery in patients with inhibitors to FVIII/IX.

Non-activated and activated prothrombin complex concentrates (PCC/aPCC) have been used successfully to treat bleeds in haemophilia patients with inhibitors, but most physicians do not consider these products as effective as factor VIII/IX (FVIII/IX) concentrates in non-inhibitor patients. Thus, surgical procedures in inhibitor patients have been performed reluctantly. We have performed 14 minor and five major surgical and invasive diagnostic procedures in eight patients with congenital haemophilia A and inhibitors and in two patients with acquired haemophilia. When a loading dose of 100 U kg-1 of FEIBA was given followed by 200 U kg-1 day-1 in three divided doses every 8 h for 3 days, and then, when the daily dose was tapered to 100-150 U kg-1, no severe or unexpected bleeding complications were observed. However, one adverse event was observed. A 69-year-old man who suffered a myocardial infarction the third postoperative day following sigmoidectomy was managed safely with opiate analgesia, nitrates and diuretics, and the continued use of FEIBA(R).

Successful thrombolysis by prolonged low-dose alteplase in catheter-directed infusion.

UNLABELLED: Catheter-directed thrombolysis is a sophisticated method in the treatment of thromboembolism with maximum effect on the thrombus and minimal systemic effect. The consequences are enhanced local thrombolysis and a reduction in general bleeding tendency, compared with systemic thrombolysis. At our institution, two children had successful thrombolysis by prolonged continuous catheter-directed low-dose alteplase. The first patient, a boy with Fontan physiology, was successfully treated for a massive pulmonary thromboembolism by catheter-directed very low-dose alteplase for five days. The second patient, who suffered from relapsing nephrotic syndrome, achieved satisfactory thrombolysis of an arterial leg thrombosis after four days of continuous catheter-directed low-dose alteplase. CONCLUSION: Although catheter-directed thrombolysis seems to be a valuable method in thrombolytic therapy, there is a lack of evidence-based recommendations concerning dosage, effect of bolus, simultaneous anticoagulation and duration of treatment for children.

Improved preservation of coagulation factors after pre-storage leukocyte depletion of whole blood.

Plasma and red blood cell quality are affected both by citrate concentration and the levels of extracellular leukocyte and platelet derived substances, accumulated during storage of blood. The effect of leukocyte filtration on the storage stability of whole blood was therefore studied in blood collected in standard CPD and 0.5CPD (CPD with half strength citrate concentration). A total of 52 units, 12 of them with reduced citrate concentration, were leukocyte-filtered with Pall( whole blood filter (WBF1 or 3). No differences in leukocyte or platelet reduction were observed with the two citrate concentrations. However, with 0.5CPD a significantly longer filtration time and increased complement activation was observed. The effect of pre-storage leukocyte filtration on the plasma quality of whole blood was therefore only studied with standard CPDA1 anticoagulant solution (normal strength citrate concentration). Leukocyte filtration did not affect the von Willebrand factor concentration, while a small reduction (7%, p=0.04) in factor VIII (FVIII) concentration was observed. During storage, however, FVIII decreased more slowly in the filtered than in the unfiltered product, and, from day two, the FVIII content was significantly higher in the filtered product (46% versus 30% at 28 days, p<0.001). Factor V (FV) demonstrated a 16% reduction (p<0.001) upon filtration, followed by an additional 8% in the next 24 h and only a 4% reduction the next 27 days, while unfiltered products demonstrated a continuous reduction to 26% at 28 days. While the beta-thromboglobulin (beta-TG) concentration significantly increased (from 836 to 2483 IU/ml, p<0.001) during leukocyte filtration, no further increase was observed during storage. In contrast, unfiltered products demonstrated an increase to 5762 IU/ml (p<0.001) at 14 days, followed by a slight, not significant, reduction. This indicates platelet activation during filtration and explains a parallel reduction in FV. Filtration induced no increase in prothrombin fragment 1+2, while a slight increase was observed in some unfiltered products after 28 days of storage.Pre-storage leukocyte depletion thus improves the coagulation factor content of plasma in stored whole blood.

CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells.

CXC-chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC-chemokines in the inflammatory interactions between oxidized (ox-) low-density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without 'traditional' risk factors and 15 carefully matched controls. Our main findings were: (a) ox-LDL stimulated the release of the CXC-chemokines interleukin (IL)-8, ENA-78 and GRO-alpha from PBMC, particularly in CAD. (b) In platelets, ox-LDL induced release of ENA-78 and, when combined with SFLLRN, also of GRO-alpha, with significantly higher response in CAD. (c) Platelet-rich plasma, especially when costimulated with ox-LDL, enhanced the release of IL-8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL-8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox-LDL, platelets and PBMC in CAD patients involving CXC-chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients.

Inflammatory imbalance between IL-10 and TNFalpha in unstable angina potential plaque stabilizing effects of IL-10.

BACKGROUND: The pathogenesis of atherosclerosis and acute coronary syndromes involves inflammation and immunological mechanisms. We hypothesized that patients with unstable angina may have an imbalance between inflammatory and anti-inflammatory cytokines. DESIGN: Plasma levels of tumour necrosis factor (TNF)alpha and interleukin (IL)-10 were analyzed in 44 patients with stable angina, 29 patients with unstable angina and 20 controls. mRNA levels of these cytokines were analyzed in peripheral blood mononuclear cells (PBMC). We also studied the in vitro effects of IL-10 in PBMC from unstable angina patients. RESULTS: Our main findings were: (1) the angina patients and particularly those with unstable disease had significantly raised TNFalpha in comparison with the controls, both at the protein and mRNA level; (2) in contrast, the levels of IL-10 were not different in the angina patients in comparison with the healthy controls, resulting in a markedly enhanced TNFalpha:IL-10 ratio, particularly in the unstable angina patients; (3) while exogenously added IL-10 markedly inhibited the release of TNFalpha, IL-8 and tissue factor as well as impairing the gelatinolytic activity and mRNA production of matrix metalloproteinase-9, it enhanced the tissue inhibitor of this metalloproteinase (i.e. TIMP-1) in PBMC from the unstable angina patients. CONCLUSION: Patients with unstable angina appear to have an imbalance between TNFalpha and IL-10, possibly favouring inflammatory net effects. IL-10 may have beneficial effects on mechanisms that are important in plaque rupture and thrombus formation.

Effects of two differently heparin-coated extracorporeal circuits on markers for brain and myocardial dysfunction.

OBJECTIVE: The two most commonly used heparin-coated systems for cardiopulmonary bypass (CPB) are the Carmeda Bio-Active Surface (CBAS) (Medtronic, Minneapolis, MN, USA) and the Duraflo II coating (Baxter Healthcare, Irvine, CA, USA). The two surfaces are technically unequal and previous experimental studies have demonstrated disparities in effects on the immune system and blood cells. However, little is known concerning the influence of the two surfaces on markers for brain and myocardial dysfunction. METHODS: Forty patients undergoing elective, primary coronary bypass grafting with CPB were prospectively randomized to either the CBAS system or the Duraflo II circuit. During and after CPB, biological markers for brain dysfunction and myocardial injury were analysed. RESULTS: Both markers for brain dysfunction S-100B and neuron-specific enolase (NSE) increased significantly during CPB (p = 0.01). The elevation during bypass correlated significantly with the duration of CPB (r = 0.39 and r = 0.38, respectively, both p < 0.02). NSE was somewhat more elevated in the Duraflo II group at the end of CPB (p = 0.01) and 5 h after CPB (p = 0.02); for S-100B, there were no intergroup differences. Also, the markers related to myocardial injury, myoglobin and creatine kinase (CK-MB) mass increased during CPB (p = 0.01), while elevation of troponin-I occurred 5 h after CPB (p = 0.01). There were no statistically significant intergroup differences. No significant correlation was seen between the release of cardiac markers and the duration of CPB. The clinical course was similar in both groups. CONCLUSIONS: Except for a slightly higher elevation of NSE at the end of CPB and 5 h after CPB in the Duraflo II group, there were no significant differences between the CBAS group and the Duraflo II group concerning markers for brain and myocardial dysfunction.

Anticoagulation intensity sufficient for haemodialysis does not prevent activation of coagulation and platelets.

BACKGROUND: A single bolus of dalteparin at the start of haemodialysis (HD) may prevent clot formation, but subclinical activation of platelets and coagulation may still occur. Consequently, the relationship between clinical clotting events and activation markers of platelets and coagulation before and during HD is of interest. METHODS: The effect of tapered doses of dalteparin during 84 HD sessions (4-4.5 h) was prospectively examined in 12 patients. Six of the patients were treated with warfarin. The initial dalteparin dose was reduced to 50% if no clotting was observed. Clinical clotting was evaluated by inspection of the air trap every hour and by inspection of the dialyser after each session. Anti-FXa activity was measured for assessment of dalteparin activity. Markers of activated plasma coagulation, (thrombin-antithrombin (TAT) and prothrombin fragment 1+2 (PF1+2)) and a marker of platelet activation (beta-thromboglobulin, beta-TG), were measured before the start of and after 3 and 4 h of dialysis. Ten pre-dialytic patients with chronic renal failure served as a control group. A total of 230 measurements of each parameter were performed. RESULTS: An anti-FXa activity above 0.4 IU/ml at the end of HD inhibits overt clot formation for 4 h. This was obtained by an intravenous dalteparin dose of about 5000 IU. TAT and PF1+2 correlated to clinical clotting episodes (r=0.50 and 0.47, P<0.001). beta-TG was not significantly correlated to clinical clotting. All parameters increased during the sessions (TAT, PF1+2, beta-TG, P<0.001). When measurements during clinical clotting episodes were disregarded, all parameters were still markedly increased. Warfarin-treated patients had lower TAT and PF1+2. Dialysis patients had higher beta-TG values than pre-dialytic patients. CONCLUSION: Despite clinically effective anticoagulation, obtained by dalteparin administration, platelets and coagulation are activated by HD, resulting in a potentially thrombophilic state. Warfarin treatment reduces clinical clot formation and subclinical activation of coagulation.

Fibrinolytic activity and postoperative salvaged untreated blood for autologous transfusion in major orthopaedic surgery.

OBJECTIVE: To evaluate the fibrinolytic activity in a closed surgical wound, in postoperatively drained blood, and during autologous transfusion. DESIGN: Prospective study. SETTING: National hospital, Norway. PATIENTS: 9 patients operated on for thoracic scoliosis. MAIN OUTCOME MEASURE: Concentrations of plasmin/antiplasmin (PAP), alpha2-antiplasmin, and D-dimers in drained, arterial, and mixed venous blood before, during, and after infusion of 10 ml/kg body weight of postoperatively drained, untreated blood. RESULTS: In drained blood the concentration of alpha2-antiplasmin was 31% of the preoperative arterial control value. Together with the increased concentrations of PAP to 18076 microg/L and D-dimers to 126 mg/L, this indicates extensive fibrinolytic activity in the closed wound. The postoperative autologous transfusion of drained, untreated blood increased the concentration of PAP from 507 to 2453 microg/L and of D-dimer from 0.7 mg/L to 15.3 mg/L in systemic blood. CONCLUSION: The systemic concentration of fibrin(ogen) degradation products, indicated by D-dimers, after recirculation of drained, untreated blood might impair coagulation. The extensive activation of plasmin might exhaust available alpha2-antiplasmin in the wound and result in postoperative rebleeding.

When should heparin preferably be administered during radiofrequency catheter ablation?

RF catheter ablation is complicated by thromboembolism in about 1% of patients. Limited knowledge exists concerning when and how to use anticoagulation or antithrombotic treatment. We studied the activation of coagulation (prothrombin fragment 1 + 2 [PF1 + 2] and D-dimer), platelets (beta-thromboglobulin [beta-TG]) and fibrinolysis (plasmin-antiplasmin complexes [PAP]) during RF ablation of accessory pathways in 30 patients. They were randomized to receive heparin (100 IU/kg, intravenously) (1) immediately after introduction of the femoral venous sheaths (group I) or (2) after the initial electrophysiological study, prior to the delivery of RF current (groups II and III). Group II additionally received saline irrigation of all femoral sheaths. After the initial bolus, 1,000 IU of heparin was supplied hourly in all groups. Within groups II and III, median plasma values of PF1 + 2 and beta-TG more than tripled (P < or = 0.007) during the diagnostic study and gradually declined during heparin administration despite RF current delivery. Median D-dimer tripled (P = 0.005) and PAP doubled (NS) before heparin administration; then both remained around the upper reference values. In the early heparin group, however, PF1 + 2, D-dimer, and PAP did not rise at all, and beta-TG showed only a slight increase towards the end of the procedure. The differences between group I versus groups II and III were statistically significant prior to the first RF current delivery (PF1 + 2, D-dimer, and beta-TG) and by the end of the procedure (PF1 + 2, D-dimer, and PAP). In conclusion, "late" heparin administration allows hemostatic activation during the initial catheterization and diagnostic study. By administering intravenous heparin immediately after introduction of the venous sheaths, hemostatic activation is significantly decreased. Saline irrigation of the venous sheaths added nothing to late heparin administration.