Calibrated automated thrombogram II: removing barriers for thrombin generation measurements.

BACKGROUND: Thrombin generation (TG) assessed by Calibrated Automated Thrombogram (CAT-I) reflects the overall capacity of plasma to generate thrombin, thus evaluating the balance between the anti- and procoagulant processes. However, with this method the calibrator curve is usually not measured until completion which has a severe impact on the calculation of the TG parameters, especially under conditions where almost all substrate is consumed. In addition, direct thrombin inhibitor (DTI) cannot be present in the calibration sample due to inhibition of the calibrator. We have developed a modified TG assay (CAT-II) and performed head-to-head comparison with the CAT-I method using the same fluorometer. Furthermore, we have compared our CAT-II method to a new automated TG instrument (ST®-Genesia) using the same calibration method. METHODS: TG was assessed with CAT-I and CAT-II using the same fulorometer and with ST®-Genesia in control plasma and plasma containing different anticoagulants (dabigatran, rivaroxaban, apixaban) and plasmas to which common interfering substances, bilirubin, hemoglobin and lipids were added. In CAT-I, calibration was against the same plasma containing calibrator in the presence of fluorogenic substrate (Z-GGR-AMC). In contrast, CAT-II method and ST®-Genesia used a standard concentration of thrombin in buffer and 7-amino-4-methylcoumarin (AMC) in a separate plasma sample for calibration. RESULTS: TG obtained from CAT-I using anticoagulant-free plasmas was lower compared with TG from CAT-II but both methods demonstrated an intra-assay variation less than 5% on all measured parameters. When comparing the two different calibration methods in the presence of different anticoagulants, a high correlation was seen in the presence of rivaroxaban and apixaban (R2 > 0.97), but not with dabigatran, a direct thrombin inhibitor. CAT-II method showed dose-dependent inhibition of TG in the presence of dabigatran, while CAT-I was not able to detect it. Both methods were able to correct for the interfering substances. CONCLUSIONS: Our results showed high similarity between the results of CAT-I and CAT-II method when it is applied in control plasmas and plasmas not inhibited with a direct thrombin inhibitor. Furthermore, both the CAT-II method and ST-Genesia using the same calibration method were able to detect the effect of all oral anticoagulants. Taken together, applying a new calibration method is a significant improvement for monitoring patients on direct thrombin inhibitors while not introducing any bias to results obtained on other types of samples.

The plasma glutamate concentration as a complementary tool to differentiate benign PET-positive lung lesions from lung cancer.

BACKGROUND: Pulmonary imaging often identifies suspicious abnormalities resulting in supplementary diagnostic procedures. This study aims to investigate whether the metabolic fingerprint of plasma allows to discriminate between patients with lung inflammation and patients with lung cancer. METHODS: Metabolic profiles of plasma from 347 controls, 269 cancer patients and 108 patients with inflammation were obtained by 1H-NMR spectroscopy. Models to discriminate between groups were trained by PLS-LDA. A test set was used for independent validation. A ROC curve was built to evaluate the diagnostic performance of potential biomarkers. RESULTS: Sensitivity, specificity, PPV and NPV of PET-CT to diagnose cancer are 96, 23, 76 and 71%. Metabolic profiles differentiate between cancer and inflammation with a sensitivity of 89%, a specificity of 87% and a MCE of 12%. Removal of the glutamate metabolite results in an increase of MCE (38%) and a decrease of both sensitivity and specificity (62%), demonstrating the importance of glutamate for discrimination. At the cut-off point 0.31 on the ROC curve, the relative glutamate concentration discriminates between cancer and inflammation with a sensitivity of 85%, a specificity of 81%, and an AUC of 0.88. PPV and NPV are 92 and 69%. In PET-positive patients with a relative glutamate level ≤ 0.31 the sensitivity to diagnose cancer reaches 100% with a PPV of 94%. In PET-negative patients, a relative glutamate level > 0.31 increases the specificity of PET from 23% to 58% and results in a high NPV of 100%. In case of discrepancy between SUVmax and the glutamate concentration, lung cancer is missed in 19% of the cases. CONCLUSION: This study indicates that the 1H-NMR-derived relative plasma concentration of glutamate allows discrimination between lung cancer and lung inflammation. A glutamate level ≤ 0.31 in PET-positive patients corresponds to the diagnosis of lung cancer with a higher specificity and PPV than PET-CT. Glutamate levels > 0.31 in patients with PET negative lung lesions is likely to correspond with inflammation. Caution is needed for patients with conflicting SUVmax values and glutamate concentrations. Confirmation is needed in a prospective study with external validation and by another analytical technique such as HPLC-MS.

Nurse telephone triage: good quality associated with appropriate decisions.

BACKGROUND: Triage at out-of-hours GP cooperatives (GPCs) is aimed at determining medical urgency and guiding decisions. Both medical knowledge and communication skills are required for this complex task. OBJECTIVE: To explore the impact of quality of consultation and estimated urgency on the appropriateness of decisions. METHODS: We performed a secondary analysis of telephone triage consultations by nurses at 29 Dutch GPCs. Consultations were taped and assessed by trained observers, using a validated instrument. Measures concerned quality of consultation, nurse-estimated urgency (four levels) and appropriateness of decisions (urgency, follow-up advice and timing). Bivariate analyses and logistic multilevel regression analyses were used to explore the impact of quality of consultation (controlling for urgency) on appropriateness of decisions. RESULTS: The sample included 6739 telephone contacts, most of which (90%) were non-urgent. The majority of decisions were appropriate (91% for urgency, 96% for follow-up advice and 95% for timing). In bivariate analyses, appropriateness of decisions was positively related to higher quality of consultation. Estimated urgency was negatively associated with quality of consultation. Logistic multilevel analysis showed that higher quality of consultation was related to a more appropriate estimation of urgency [odds ratio (OR) = 1.82; 95% confidence interval (CI): 1.69-1.95], follow-up advice (OR = 2.69; 95% CI: 2.41-3.01) and timing of decisions (OR = 2.41; 95% CI: 2.20-2.63). CONCLUSIONS: High quality of consultation has a positive, but small, impact on the appropriateness of decisions. Quality of consultation needs to be targeted in training and support of triage nurses, especially when it concerns highly urgent contacts.

Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury.

[Self-referrals at Emergency Care Access Points and triage by General Practitioner Cooperatives]. / Zelfmelders op de spoedpost bij triage door de huisartsenpost.

OBJECTIVE: There is a trend for General Practitioner Cooperatives (GPCs) to co-locate with emergency departments (EDs) of hospitals at Emergency Care Access Points (ECAPs), where the GPCs generally conduct triage and treat a large part of self-referrals who would have gone to the ED by themselves in the past. We have examined patient and care characteristics of self-referrals at ECAPs where triage was conducted by GPCs, also to determine the percentage of self-referrals being referred to the ED. DESIGN: Retrospective cross-sectional observational study. METHOD: Descriptive analyses of routine registration data from self-referrals of five ECAPs (n = 20.451). Patient age, gender, arrival time, urgency, diagnosis and referral were analysed. RESULTS: Of the self-referrals, 57.9% was male and the mean age was 32.7 years. The number of self-referrals per hour was highest during weekends, particularly between 11 a.m. and 5 p.m. On weekdays, there was a peak between 5 and 9 p.m. Self-referrals were mostly assigned a low-urgency grade (35.7% - U4 or U5) or a mid-urgency grade (49% - U3). Almost half of the self-referrals had trauma of the locomotor system (28%) or the skin (27.3%). In total, 23% of the patients was referred to the ED. CONCLUSION: Self-referred patients at GPCs are typically young, male and have low- to mid-urgency trauma-related problems. Many self-referrals present themselves on weekend days or early weekday evenings. Over three quarters of these patients can be treated by the GPCs, without referral to the ED. This reduces the workload at the ED.

Patients either contacting a general practice cooperative or accident and emergency department out of hours: a comparison.

INTRODUCTION: Lack of collaboration between general practice (GP) cooperatives and accident and emergency (A&E) departments and many self referrals may lead to inefficient out-of-hours care. METHODS: We retrospectively analysed the records of all patients contacting the GP cooperative and all patients self referring to the A&E department out of hours in a region in the Netherlands. RESULTS: 258 patients contacted the GP cooperative and 43 self referred to the A&E department per 1000 patients per year. A wide range of problems were seen in the GP cooperative, mainly related to infections (26.2%). The A&E department had a smaller range of problems, mainly related to trauma (66.1%). Relatively few urgent problems were seen in the GP cooperative (4.6%) or for self referrals in the A&E department (6.1%). Women, children, the elderly, and rural patients chose the GP cooperative significantly more often, as did men and patients with less urgent complaints, infections, and heart and airway problems. DISCUSSION: The contact frequency of self referrals to the A&E department is much lower than that at the GP cooperative. Care is complementary: the A&E department focuses on trauma while the GP cooperative deals with a wide range of problems. The self referrals concern mostly minor, non-urgent problems and can generally be treated by the general practitioner, by a nurse, or by advice over the telephone, particularly in the case of optimal collaboration in an integrated care facility of GP cooperatives and A&E departments with one access point to medical care for all patients.

[Practice assistant sometimes misses urgent request for help: telephone triage in general practice]. / Doktersassistent mist soms hoogurgente hulpvraag.

OBJECTIVE: To assess the organisation and appropriateness of telephone triage in general practices in the Netherlands. DESIGN: Cross-sectional observational study. METHODS: Via e-mail we invited all members of the Dutch Association of practice assistants to complete an online survey. The questionnaire included questions about practice assistants' background characteristics and the practices' triage organisation. Furthermore, they were asked to assess the indicated type of care for a number of fictive case scenarios involving a variety of health problems and levels of urgency. To determine the appropriateness of the respondents' assessments, each was compared to a reference standard agreed by experts. In addition, the association between practice assistants' background characteristics and organizational setup of the triage organisation with the appropriateness of triage was examined. RESULTS: The response rate was 41.1% (N=973). The required care was assessed appropriately in 63.6% of the cases, over-estimated in 19.3% and under-estimated in 17.1% of cases. The sensitivity of identifying patients with a highly urgent problem was 76.7%, whereas the specificity was 94.0%. The appropriateness of the assessments of the required care was higher for more experienced assistants and assistants with regular daily work meetings with the GP. Triage training, use of a triage tool and authorization of advice provision were not associated with appropriateness of triage. CONCLUSION: Triage by practice assistants in general practices is efficient, but potentially unsafe in highly urgent cases. It is therefore important to train practice assistants in the identification of highly urgent cases.

Production of thrombin as a probe for mixing of phospholipids in membranes on solid supports.

Phospholipid-covered solid supports have been used successfully as model membranes in studies on blood coagulation and other research fields. In order to produce such membranes, simple exposure of the support to suspensions of phospholipid vesicles was recently introduced, but questions have remained about the process of vesicle adherence to the surface and the physico-chemical properties of the resulting membranes. Using a new technique, mixing of phospholipids in such membranes was demonstrated. A rotating, hydrophilic, silicon disc was exposed in a two-step procedure to vesicles prepared from mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC). Factor Xa, factor Va and prothrombin were added and the transport-limited production rate of thrombin was measured. For low surface coverage with 40% DOPS/60% DOPC, a much higher conversion rate was found if, prior to addition of coagulation factors, excess DOPC vesicles were added to fill up vacant surface area. It is concluded that DOPS is spread over the entire surface and that confluent bilayers are formed. The presented technique may also be used to measure lateral diffusion constants.

Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry.

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant Kd = 140 +/- 27 nM (mean +/- S.E.) and maximal surface concentration gamma max = (8.9 +/- 0.8) x 10(-3) mole of protein per mole of lipid, were obtained. For factor Xa, these values were Kd = 49.6 +/- 6.3 nM and gamma max = (23.0 +/- 1.4) x 10(-3) mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.

Initiating and potentiating role of platelets in tissue factor-induced thrombin generation in the presence of plasma: subject-dependent variation in thrombogram characteristics.

The hemostatic activity of plasma is determined by platelet activation and coagulation, which processes are mutually stimulatory. We studied this interaction by measuring the cleavage of fluorescent thrombin substrate in platelet-rich plasma (PRP), using the calibrated thrombogram method. In freshly isolated human plasma, thrombin formation triggered by tissue factor was fully dependent on the presence of platelets. It was abolished by annexin A5, indicating dependence on phosphatidylserine (PS) exposure at activated platelets. Comparison of plasmas from various subjects showed considerable interindividual variation in total amount of thrombin generation, regardless of whether platelets or PS-containing phospholipids were present. Integrin alphaIIbbeta3 antagonists and ADP receptor blockage, but not aspirin, decreased the rate of thrombin generation (thrombin peak level) and extended the time of onset. Platelet inhibition with cAMP-elevating agents decreased the thrombin-forming rate, but surprisingly shortened the onset time. Stimulation of platelets with agonists of Gi/q-coupled receptors and, to a larger extent, with collagen or Ca2+-ionophore increased the rate of thrombin generation and shortened its onset. In PRP from donors with low and high generation, platelet inhibitors and activators were similarly effective. Taken together, these results indicate that, in tissue factor-triggered PRP, PS exposure on activated platelets regulates both onset and rate of thrombin generation. However, coagulant activity rather than platelet activation determines the total amount of thrombin formed, i.e. the endogenous thrombin potential. Thus, kinetics of thrombin generation in PRP are controlled by platelet inhibitors and agonists, but the process is restricted in amount by the subject-dependent variation in coagulation.

Regulation of the procoagulant response to arterial injury.

The last few years have provided increasing evidence to support a major role for TF in the initiation and propagation of thrombosis after acute arterial injury. Although thrombotic occlusion occurs in a small minority of patients undergoing acute coronary interventions or bypass surgery, mural thrombi are likely to be present in almost all cases. These thrombi may stimulate SMC and promote the development of intimal hyperplasia and luminal narrowing. The use of inhibitors of TF and factor VIIa, therefore, may not only be valuable for inhibiting thrombus formation associated with acute arterial interventions, but may also have benefit in attenuating intimal hyperplasia. Although this paper focuses on the role of TF in establishing a procoagulant state after arterial injury, the fibrinolytic system undoubtedly plays a role in balancing the effects of increased TF production in the arterial wall. This is underscored by the success of activators of fibrinolysis (tissue plasminogen activator, streptokinase, urokinase) in revascularization in the setting of acute myocardial infarction and is reviewed elsewhere. Likewise, local regulation of TFPI in the atherosclerotic plaque and injured vessel wall may be important in attenuating the effects of increased TF synthesis and accumulation. It has been assumed that the primary source of active TF after arterial injury is either SMC or invading macrophages and that active TF is anchored to the surface of these cells. Recent data have suggested that the majority of cell-associated TF is either encrypted on the cell surface or present in an intracellular pool. Arterial injury may, therefore, involve the de-encryption of surface TF or the release of intracellular TF. In addition, active vascular TF may be present in microparticles that are not anchored to the arterial wall and may be washed into the circulation. The procoagulant state may be further accentuated by the accumulation of bloodborne TF at sites of arterial injury and in developing thrombi. This TF is likely to arise from circulating leukocytes, including neutrophils and monocytes. These studies suggest that the cellular processing of TF may be an important target for inhibiting thrombotic complications associated with arterial injury and acute coronary events.

The thrombogram: monitoring thrombin generation in platelet-rich plasma.

A method is described in which thrombin activity in clotting plasma can be monitored through the continuous measurement of the fluorescent split-product of the substrate Z-Gly-Gly-Arg-AMC. The signal is not impaired by turbidity; therefore proper measurement is not disturbed by the occurrence of a clot or the presence of platelets and direct measurement in platelet rich plasma is possible.

Tissue factor in the pathogenesis of atherosclerosis.

TF antigen and activity are found in abundance in human atherosclerotic plaques, particularly in the lipid-rich core. TF is also readily induced in the arterial wall by balloon injury and accumulates in the resulting neointima. In chronic atherosclerosis, the macrophage is likely to be the major source of TF within the plaque. TF accumulates as an early event associated with the migration of monocytes to the vessel wall in response to chemoattractants, such as MCP-1, and their differentiation into macrophages. As SMC become activated in the developing plaque, they provide a second source of TF. Macrophages and SMC accumulate lipid and become foam cells, ultimately degenerating into a necrotic core rich in TF. Spontaneous plaque rupture or acute interventions expose active TF in the core to circulating blood, triggering thrombosis. In acute arterial injury, SMC appear to be the chief source of TF. In normal vessels, the induction of TF in the medial SMC is not sufficient to generate fibrin, presumably because the TF is not readily accessible on the luminal surface. In contrast, endothelial denudation of previously injured arteries may expose intimal TF to circulating blood, resulting in rapid fibrin deposition. In advanced human atherosclerosis, it is likely that even in areas that do not contain "unstable" or "stable" plaques, the vessel wall is not normal and more closely resembles that of a previously injured artery possessing an active intima. Interventions, such as balloon angioplasty, coronary atherectomy, or stent placement may expose intimal TF, leading to fibrin deposition. As the initiator of coagulation, TF is a potential target for inhibiting the thrombotic complications of atherosclerosis. TFPI (reviewed in 52) is currently under clinical investigation as an anticoagulant and its effects on intimal hyperplasia in animal models are being studied. Direct factor Xa inhibitors, such as tick anticoagulant peptide (TAP) and leech anticoagulant peptide (ATS), are also under investigation (53-54). Finally, the recent crystallization of TF (55) and the TF:VIIa (56) should provide important new insights into the design of molecules for directly inhibiting TF.

Intimal tissue factor activity is released from the arterial wall after injury.

Tissue factor (TF), the initiator of coagulation, has been implicated as a critical mediator of arterial thrombosis. Previous studies have demonstrated that TF is rapidly induced in the normal rodent arterial wall by balloon injury, but is not associated with fibrin deposition. A second injury, however, performed 10-14 days after the first, is followed by small platelet-fibrin microthrombi. This study was undertaken to better localize active TF in balloon-injured rat arteries and to explore possible mechanisms underlying the apparent discrepancy between injury-induced TF expression and the lack of large platelet-fibrin thrombi. By immunohistochemistry, TF antigen was first detected in the media 24 h after injury to rat aortas, and subsequently accumulated in the neointima. Using an ex vivo flow chamber, no TF activity (Factor Xa generation) was found on the luminal surface of normal or injured aortas. Wiping the luminal surface with a cotton swab exposed TF activity in all vessels; levels were increased approximately 3-fold in arteries containing a neointima. The exposed TF activity was rapidly washed into the perfusate, rendering the luminal surface inactive. The loss of luminal TF into the circulation may attenuate thrombosis at sites of arterial injury.

Some thoughts about localization and expression of tissue factor.

It is likely that tissue factor (TF) evolved as a haemostatic protein and, as such, it is highly concentrated in vascular tissue. Most cell surface TF is latent and simple exposure of the cell surface to circulating procoagulant proteins is not sufficient to trigger coagulation. Recently, it has been shown that an intracellular pool of TF accumulates after stimulation of vascular smooth muscle cells with growth factors. We have estimated that 20% of cellular TF is available on the surface, 30% is intracellular and 50% is latent. Since the bulk of cell surface TF is latent, staining vessels for TF does not accurately reflect their haemostatic and thrombogenic potential. It has long been thought that, in vivo, initiation of haemostasis requires only disruption of the vascular wall. We have detected vesicular TF in arterial sections raising the possibility that this pool of TF initiates thrombosis and possibly haemostasis. Much progress has been made in investigating the role and mode of action of TF, but fundamental questions remain to be answered.

[The practice guideline 'Pregnancy and puerperium' (first revision) from the Dutch College of General Practitioners; a response from the perspective of general practice medicine]. / De standaard 'Zwangerschap en kraamperiode' (eerste herziening) van het Nederlands Huisartsen Genootschap; reactie vanuit de huisartsgeneeskunde.

The first revision of the Dutch College of General Practitioners' practice guideline about pregnancy and puerperium does not significantly differ from the first edition. The guideline is extensive, is well-worth reading and supports daily practice. There is a greater emphasis on the importance of cooperation and differentiation in primary care (midwifes and general practitioners). During the last decade many general practitioners stopped doing home deliveries and have therefore lost their experience in obstetric care and pathology. The guideline describes the general practitioner's tasks as a preconception counsellor, a professional expert on illnesses during pregnancy and after the delivery, and as the doctor of the newborn baby. It will hopefully stimulate a revived interest of and involvement in pregnancy and post-partum care among general practitioners.

The impact on emergency department utilization and patient flows after integrating with a general practitioner cooperative: an observational study.

Introduction. A new model, an emergency care access point (ECAP) for after-hours emergency care, is emerging in The Netherlands. This study assessed the effect on emergency department (ED) utilization and patient flows. Methods. Routinely recorded clinical ED patient data, covering a six-year period, was collected. Segmented regression analysis was used to analyze after-hours changes over time. Results. 59.182 patients attended the ED before the start of the ECAP and 51.513 patients after, a decrease of 13%. Self-referred ED patients decreased 99.5% (OR 0.003; 95% CI 0.002-0.004). Referred patients increased by 213.4% and ED hospital admissions increased by 20.2%. A planned outpatient follow-up increased by 5.8% (OR 1.968 95% CI 1.870-2.071). The latter changed from fewer contacts to more contacts (OR 1.015 95% CI 1.013-1.017). Consultations at the regional genereral practitioner cooperative (GPC) increased by 26.0% (183.782 versus 232.246). Conclusion. ECAP implementation resulted in a decrease in ED utilization, a near absence of self-referring patients, and a higher probability of hospital admission and clinical follow-up. This suggests either an increase of ED patients with a higher acuity or a lower threshold of admitting referred patients compared to self-referred patients. Overall, increased collaboration with after-hours primary care and emergency care seemed to optimize ED utilization.

Antidote strategies to reverse anticoagulation with TB-402, a long-acting partial inhibitor of factor VIII.

BACKGROUND: TB-402 is a partially inhibiting antibody of factor VIII that is under development as a long-acting anticoagulant. PATIENTS AND METHODS: The reversibility of FVIII inhibition by TB-402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma-derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg(-1) rhFVIII 48 h after a single dose of 620 µg kg(-1) TB-402. TB-402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. RESULTS: In spiked samples, TB-402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 µg mL(-1) TB-402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB-402. The inhibitory effect of TB-402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half-life (t(1/2) ) of TB-402 was 14.2 days. TB-402 lowered the endogenous thrombin potential by 23% for ~ 35 days. Infusion of 35 IU kg(-1) rhFVIII had a marginal effect, whereas 70 IU kg(-1) rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ~ 3 h. CONCLUSIONS: TB-402 resulted in a stable long-term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB-402 temporarily, and may be effective antidotes for future clinical practice.

Tissue factor on the loose.

The enzymatic complex of tissue factor (TF) and the blood coagulation factor VIIa is generally considered to be the initiator of coagulation. Coagulation that occurs at the site of luminal injury to an artery is, along with platelet deposition, the cause of arterial thrombosis, which is the leading cause of death in Western society. Under pathological conditions the intima, the neointima and the atherosclerotic plaque contain active TF. Therefore the initiation of thrombosis is believed to be due to TF present in the wall of the pathologically changed artery. This classical view of thrombosis has been challenged. In this article we review the evidence for the presence of TF activity in various tissues outside the vessel wall, in extracellular form, in encrypted form, and even in plasma. We found TF expression in a variety of cells in culture after growth factor or cytokine stimulation. This TF was often also present in the extracellular matrix, and in addition we found latent TF on the outside of unbroken smooth muscle cells. Freeze-thawing the cells or detergent lysis could activate this TF. We also found TF activity in native whole blood and in plasma. Inhibition of this circulating TF prevented formation of thrombi on collagen-coated glass slides in an ex vivo perfusion system. Furthermore, in a thrombosis model in which rat aorta was injured, TF was found on the intimal surface of the injured aorta. TF activity was measured in a flow chamber, and it was shown that all measurable activity was extracellular. We conclude that blood-borne TF plays a major role in thrombosis. Encryption of TF present in circulation could be a mechanism that prevents thrombosis. Alternatively, circulating TF may be active but below the threshold required for the initiation of blood coagulation.