Overall Fibrinolytic Potential Research Articles

Acute effects of high intensity interval training versus moderate intensity continuous training on haemostasis in patients with coronary artery disease

Exercise training is associated with an acute net increase in coagulation, which may increase the risk of atherothrombosis in coronary artery disease (CAD) patients. We sought to compare the acute haemostatic effects of a bout of moderate-intensity continuous (MICT) and high-intensity interval training (HIIT) in patients with CAD. Patients after a recent myocardial infarction were randomized into a HIIT or MICT session of exercise training on a stationary bike. Blood was sampled at baseline, after the exercise bout and after a one-hour resting period. We measured overall haemostatic potential (OHP), overall coagulation potential (OCP), fibrinogen, D-dimer and von Willebrand factor (vWF) and calculated overall fibrinolytic potential (OFP). Linear mixed models for repeated measures were constructed to assess the treatment effect. A total of 117 patients were included. OCP, OHP, fibrinogen, D-dimer and vWF significantly increased after exercise and returned to baseline after a one-hour rest, OFP decreased after exercise and returned to baseline levels after a one-hour rest. Linear mixed models showed a significant difference between HIIT and MICT in fibrinogen (p 0.043) and D-dimer (p 0.042). Our study has shown that an exercise bout is associated with a transient procoagulant state in patients with CAD, with similar exercise-induced haemostatic changes for HIIT and MICT.

Extracellular vesicles in plasma and cerebrospinal fluid in patients with COVID-19 and neurological symptoms.

Increased levels of extracellular vesicles (EVs) are associated with haemostatic disturbances in various clinical settings. However, their role in COVID-19 patients is still not fully clear. In the present study we investigated EVs in plasma from patients with COVID-19 and neurological symptoms in relation to the activation of coagulation. Nineteen COVID-19 patients with neurological symptoms and twenty-three aged-matched healthy individuals were included. Global coagulation assays were performed and levels of EVs were determined by flow-cytometry in plasma and cerebrospinal fluid (CSF). A procoagulant state characterized by significantly increased overall coagulation- (OCP) and overall haemostatic potential (OHP), diminished overall fibrinolytic potential (OFP) together with a denser fibrin structure was found in patients with COVID-19. Flow cytometry revealed elevated levels of plasma circulating EVs derived from neutrophils (MPO+) and platelets (CD61+), as well as EVs expressing phosphatidylserine (PS+) and complement component C5b-9 (TCC+) in patients with COVID-19 compared with controls. The concentrations of PS+, CD61+ and TCC+ EVs were positively correlated with OCP and OHP in COVID-19 patients. Moreover, we identified CD61+, MPO+ and endothelial cell-derived EVs, as well as EVs exposing PS and TCC in the CSF of patients suffering from neurological symptoms during COVID-19. The unique finding in this study was the presence of EVs in the CSF of COVID-19 patients with neurologic manifestations as well as higher expression of complement protein on circulating plasma EVs. EVs may indicate blood-brain barrier damage during SARS-COV-2 infection.

The Correlation between the Triglyceride-Glucose Index and Coagulation Markers in Patients with Recent Acute Myocardial Infarction.

Background Metabolic abnormalities and hypercoagulability seem to have an important predictive role in patients with coronary artery disease (CAD). The triglyceride-glucose (TyG) index has emerged as a good marker for metabolic syndrome with predictive value for cardiovascular events. Overall haemostatic potential (OHP) is a reliable global haemostatic essay to identify hypercoagulability in CAD patients. The aim of our study was therefore to evaluate a possible correlation between the TyG index and haemostatic derangements in patients with CAD. Methods Consecutive patients referred for the first follow-up visit after acute myocardial infarction between December 1, 2018, and March 31, 2020, and did not meet exclusion criteria were included. We determined OHP, overall coagulation potential (OCP), overall fibrinolytic potential (OFP), fibrinogen, D-dimer, and von Willebrand factor from peripheral blood samples. The TyG index was calculated with the previously described and validated formula. Linear regression models were constructed for the multivariate analysis. Results A total of 117 patients (mean age 56 ± 10 years, 20% women) were included. A correlation was found between TyG index and OCP (r = 0.229, p = 0.026), TyG index and OHP (r = 0.202, p = 0.050), and TyG index and fibrinogen (r = 0.271, p = 0.005). In the multivariate model which accounted for sex, age, and BMI, the correlation between TyG index and OCP (R2 0.108; ANOVA for regression p = 0.035; beta 2.08 [0.79-4.01], p = 0.042) and between TyG index and fibrinogen (R2 0.11; ANOVA for regression p = 0.015; beta 0.35 [0.08-0.62], p = 0.012) emerged as statistically significant. Conclusion The TyG index, a marker of metabolic syndrome, has a strong correlation with a hypercoagulability state in CAD, as determined by the OCP and higher fibrinogen levels. Our findings suggest that metabolic syndrome may be an important driver of atherothrombotic risk in patients with CAD.

Association between Lipoprotein Subfractions, Hemostatic Potentials, and Coronary Atherosclerosis.

Background Dyslipidemias are associated with atherosclerotic plaque formation and a prothrombotic state, thus increasing the risk of both atherosclerotic vascular disease and atherothrombotic adverse events. We sought to explore the association between lipoprotein subfractions, overall hemostasis, and coronary calcifications in individuals at intermediate cardiovascular risk. Methods Consecutive statin-naive individuals at intermediate cardiovascular risk referred for coronary artery calcium score (CACS) scanning were included. CACS was assessed using a 128-slice dual-source CT scanner. Traditional lipid profile, high-density lipoprotein (HDL) subfractions 2 and 3, and small dense low-density lipoproteins (sdLDL) were measured with commercially available assays. Overall hemostatic (OHP) and coagulation potentials (OCP) were measured spectrophotometrically, using fibrin aggregation curves after exposure to thrombin and recombinant tissue-type plasminogen activator, respectively. Overall fibrinolytic potential (OFP) was calculated as a difference between the two areas under curves. Results We included 160 patients (median age 63 (interquartile range (IQR), 56-71 years, 52% women, and median CACS 8, IQR 0-173 Agatston units). HDL3 levels—but not sdLDL or hemostatic potentials—were significantly associated with CACS zero, even after adjusting for age, sex, arterial hypertension, dyslipidemia, diabetes, and smoking history (OR 0.980 (0.962-0.999), p = 0.034). HDL3 was also significantly associated with OCP (r = −0.232, p adjusted for age and sex 0.037). Conclusions In patients at intermediate cardiovascular risk, HDL3 is associated with both subclinical atherosclerosis and overall coagulation. Our findings are in line with studies reporting on an inverse relationship between HDL3 and atherosclerosis and provide one possible mechanistic explanation for the association between novel lipid biomarkers and coagulation derangements.

OP0059 PROFOUND ANTICOAGULANT EFFECTS OF INITIAL ANTIRHEUMATIC TREATMENTS IN EARLY RHEUMATOID ARTHRITIS PATIENTS: A NORD-STAR SPIN-OFF STUDY

BackgroundPatients with rheumatoid arthritis (RA) are at an increased risk of venous thromboembolism. Thus far, there have not been any comparative studies investigating the effects of initial antirheumatic treatments in (very) early RA patients.ObjectivesTo assess the effects of different initial treatments on hemostatic parameters in patients with early RA.MethodsNORD-STAR is an international, multicentre, open-label, assessor-blinded, phase 4 study where patients with newly diagnosed RA started methotrexate (MTX) and were randomised 1:1:1:1 to a) conventional treatment (either prednisolone tapered to 5mg/day, or sulfasalazine combined with hydroxychloroquine and intra-articular corticosteroids), b) certolizumab pegol, c) abatacept, d) tocilizumab1. This study is a spin-off from the main NORD-STAR study extensively investigating hemostatic system in 24 per protocol consecutive Dutch participants at baseline, 12 weeks and 24 weeks after the start of the treatment. Statistical analysis was done using paired samples t-test in SPSS version 28.ResultsThe mean age of investigated patients was 51.8 (± 12.7) years and 58.3% were female. At baseline patients had an average DAS28 score of 4.6 (± 0.9) and had elevated levels of investigated coagulation biomarkers: Factor 1 + 2, fibrinogen, D-dimer and parameters of the two global hemostatic assays, i.e. endogenous thrombin potential (ETP) and overall hemostasis potential (OHP). These biomarkers decreased significantly at 12 and 24 weeks in patients in all groups (Table 1). Overall fibrinolytic potential (OFP) was decreased and clot lysis time (CLT) was prolonged at baseline, demonstrating impaired fibrinolytic activity in early RA. The reduction of coagulation parameters was significantly higher in biological treatment arms in comparison to the standard MTX treatment arm. In addition, tocilizumab was more effective compared to certolizumab and abatacept, (Figure 1), which was expected considering the direct inhibitory effect of this drug on the IL-6 synthesis and consequently the coagulation activation as well. After 24 weeks of treatment with methotrexate and tocilizumab, the average fibrinogen of patients was reduced by 63% vs 31% and 36% in the certolizumab and abatacept groups, respectively. The changes in DAS-28 and the changes in fibrinogen had a correlation of 0.385 which did not reach statistical significance.Table 1.Measurements are marked with * if p<0.05, ** if p<0.01 and *** if p<0.001BaselineW12W24Factor 1 + 2 (pmol/L)270.25 (149.4)190.36 (108.6)**179.52 (85.3)***Fibrinogen (g/L)4.64 (1.5)3.61 (1.6)**2.63 (1.2)***D-dimer (mg/L)2.17 (3.0)0.33 (0.23)**0.29 (0.2)**OHP (Abs-sum)157.38 (64.9)120.62 (68.7)*100.49 (53.8)***OCP (Abs-sum)369.52 (58.8)305.04 (101.7)*275.91 (83.1)***OFP (%)57.97 (13.1)63.20 (12.7)*65.25 (11.4)***Lag time (s)304.5 (71.1)306.8 (71.8)312.7 (65.4)Slope0.07 (0.02)0.066 (0.03)0.094 (0.12)Max Abs1.17 (0.3)1.00 (0.4)*0.91 (0.3)**CLT (s)1405 (356)1317 (377)1231 (320)**ETP (nM*min)1480 (471)1395 (395)*1337 (429)*Peak (nM)231 (78)223 (68)223 (74)Lagtime (min)4.06 (2.1)3.28 (1.2)**2.87 (1.0)***ttPeak (min)7.40 (2.2)6.61 (1.5)*6.13 (1.4)**Figure 1.ConclusionOur results indicate an enhanced coagulation and fibrinolytic impairment in newly diagnosed RA patients. Effective antirheumatic treatments reduce this hemostatic imbalance, with significantly more pronounced effects of biologic drugs compared to conventional (MTX+glucocorticoids) treatment.

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis.

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0–25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a+), leucocyte (CD45+), monocyte (CD14+), and endothelial (CD144+)-derived EVs; and EVs-expressing tissue factor (CD142+), P-selectin (CD62P+), and E-selectin (CD62E+) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a+, CD142+, CD45+, CD14+, and CD62P+ EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a+, CD14+, and CD62P+ EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a+ EVs and OCP as well as between the levels of PS+, CD42a+, and CD62P+EVs and OHP. The levels of PS+, CD42a+, CD14+, CD62P+, and CD62E+ EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease.

Venous thromboembolism in anti-neutrophil cytoplasmic antibody-associated vasculitis: an underlying prothrombotic condition?

ObjectivesWe investigated the incidence and potential underlying risk factors of venous thromboembolism (VTE) in patients with AAV. We assessed haemostatic disturbances and factors that might contribute to the risk of development of VTE.MethodsANCA-positive AAV patients (n = 187) were included. Previously identified risk factors for VTE and current medication were retrieved from the medical records. We assessed haemostasis using different methods [endogenous thrombin potential (ETP), overall haemostatic potential (OHP), overall coagulation potential (OCP) and overall fibrinolysis potential (OFP)] in patients with active AAV (n = 19), inactive AAV (n = 15) and healthy controls (n = 15).ResultsTwenty-eight VTEs occurred in 24 patients over a total follow-up time of 1020 person-years. A majority of VTEs occurred within the first year after diagnosis. Old age (P < 0.01), ongoing prednisolone treatment and recent rituximab administration were more common in the VTE group (P < 0.05 for all). ETP and OHP were significantly increased and OFP significantly decreased in plasma from active compared with inactive AAV patients (P < 0.05, P < 0.01 and P < 0.05, respectively) and healthy controls (P < 0.001). We could not confirm previously reported risk factors for VTE development.ConclusionA high prevalence of VTE in AAV patients was seen within the first year after diagnosis, suggesting that disease activity contributes to development of VTE. Old age and concurrent treatment should also be taken into account when estimating VTE risk. The results also indicate disturbances in the haemostatic balance towards pro-thrombotic conditions in AAV patients, where ETP and OHP might be useful markers for identifying patients at high risk.

Assessment of hemostatic disturbances in women with established rheumatoid arthritis

ObjectivesThis study was aimed to assess hemostatic disturbances in female patients with established rheumatoid arthritis (RA) in relation to menopausal status and disease activity.MethodNinety women were included in the study, 42 patients and 48 age-matched healthy controls. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. Two global hemostatic assays were employed, namely endogenous thrombin potential (ETP) and overall hemostasis potential (OHP). The parameters of the ETP assay (ETP, C-max, t-lag, t-max) and OHP assay (overall coagulation potential (OCP) and overall fibrinolytic potential (OFP)) were assessed. Moreover, the parameters of the fibrin clot (lag time, Max Abs, and slope) were measured by clot turbidity and scanning electron microscopy (SEM). Both patients and controls were divided into four subgroups according to menopause status.ResultsThe premenopausal controls differed significantly from all other subgroups in terms of diminished levels of ETP (p = 0.02), C-max (p = 0.01), OCP (p = 0.02), OHP (p = 0.001), and Max Abs (p = 0.008), while OFP (p = 0.0001) was increased. This tendency was not seen in the premenopausal RA patients compared with the postmenopausal RA patients. SEM images showed denser clots composed of thinner fibers in samples from RA patients. The disease activity measured by DAS28 correlated with OCP and OHP (r = 0.54; p = 0.001 and r = 0.44; p = 0.003, respectively) indicating persistent hypercoagulable condition in the whole group of RA patients.ConclusionsOur results point towards coagulation activation in premenopausal women with established RA. The patients were well characterized, which enabled assessment in a real-life setting.Key Points• Extensive assessment points towards persistent coagulation activation in premenopausal women with established rheumatoid arthritis.• Impaired thrombin generation and fibrin formation are associated with menopause in healthy women, while rheumatoid arthritis closes the gap within patients regarding menopause.• Fibrin morphology is unfavorably altered and fibrinolysis is decreased in patients with established rheumatoid arthritis.• Increased activity of thrombin activatable fibrinolysis inhibitor (TAFI) may contribute to impaired fibrinolysis in patients with rheumatoid arthritis.

Increased Coagulation and Decreased Fibrinolysis as Measured with Overall Hemostatic Potential Are Dependent on BMI and Not Associated with PCOS.

Overall hemostatic potential (OHP) captures all factors that affect coagulation and fibrinolysis cascade. It has not yet been assessed in polycystic ovary syndrome (PCOS). The aim of the study was to identify the relationship of OHP with a syndrome per se and body mass index (BMI). In 90 women with PCOS aged 30.9 ± 8.1 years (50 obese, 13 overweight, and 27 lean) and 21 healthy age-matched controls (11 obese and 10 lean), OHP with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) was determined spectrophotometrically. OFP was calculated. OHP increased with BMI in PCOS (9.6 ± 2.3 in lean, 12.5 ± 5.1 in overweight, and 15.5 ± 3.8 Abs-sum in obese) and in controls (9.1 ± 1.0 in lean and 17.3 ± 4.6 Abs-sum in obese). There was significant difference between lean and obese PCOS (P < 0.001) and between lean and obese controls (P < 0.001). OCP also increased with BMI in PCOS (P < 0.001 for lean vs. obese) and in controls (P < 0.001 for lean vs. obese). OFP decreased with BMI in PCOS (P < 0.001 for obese vs. overweight vs. lean) and in controls (P < 0.001 for obese vs. lean). OHP in healthy obese and obese PCOS did not differ significantly, while OHP for healthy obese was increased in comparison to overweight and lean PCOS (P < 0.001). PCOS was not associated with increased OHP compared with BMI and age-matched controls. However, increase in OHP was positively associated with BMI in PCOS and healthy women.

Persistent global hypercoagulability in long-term survivors of acute pulmonary embolism.

Hypercoagulable and/or hypofibrinolytic states are risk factors for venous thromboembolism (VTE) including acute pulmonary embolism. Current screening for thrombophilia is targeted towards identifying a specific defect and guidelines recommend a population-based rather than individualized strategy for anticoagulation treatment. We investigated whether there is a global hypercoagulable state in long-term survivors of pulmonary embolism no longer receiving therapeutic anticoagulation utilizing the overall haemostatic potential (OHP) assay, which assesses overall coagulation potential (OCP), OHP and overall fibrinolytic potential (OFP). Long-term survivors of acute pulmonary embolism were identified from a local registry and OHP assays were performed and compared with age and sex-matched controls without pulmonary embolism. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption 405 nm) after addition of tissue factor and tissue plasminogen activator to plasma. OHP assays were performed in 67 long-term survivors of single pulmonary embolism (7.9 ± 1.4 years after pulmonary embolism) and 20 age (61.7 ± 11.2 vs 56.6 ± 6.4 years, P = 0.06) and sex (P = 0.45)-matched controls. Survivors of pulmonary embolism were more hypercoagulable as reflected by significantly higher OCP (56.4 ± 13.0 vs 49.9 ± 6.9, P = 0.03) and had impaired fibrinolysis with higher OHP (12.6 ± 7.0 vs 5.9 ± 2.0, P < 0.001) and lower OFP (78.1 ± 9.4 vs 88.2 ± 2.9, P < 0.001) compared with controls. Importantly, these abnormalities in overall coagulation were independently predicted by levels of fibrinogen, platelet count, shortened activated partial thromboplastin time and inflammatory markers suggesting a multifactorial cause. Long-term survivors of pulmonary embolism demonstrate enhanced global coagulation and reduced fibrinolytic potential. Assessment of global coagulation may provide new insights into the aggregate effects of multiple prothombotic factors and long-term risk of VTE recurrence.

Enhanced thrombin generation in women with a history of oral contraception-related venous thrombosis

In women who suffer venous thrombosis (VT) during oral contraceptive (OC) use, a transient risk factor (OC) is removed during the acute event, while most co-existing forms of thrombophilia persist and presumably continue to maintain hypercoagulability. The aim of this study was to establish if hypercoagulability persists long after OC-related VT and if it could be attributed to thrombophilia. 60 women (age 33.0 ± 8.5 years) were investigated 5 - 64 (median 33) months after OC-related VT (patients) and compared to 63 apparently healthy women (controls). All women were tested for thrombophilia, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, P-selectin and C-reactive protein. Thrombin generation was measured by Technothrombin® TGA assay. Overall haemostasis potential (OHP) assay with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) as supplementary parameters were measured by repeated fibrin formation and degradation registration. In patients increased endogenous thrombin potential (4,205 ± 440 nM x min vs 4,015 ± 421 nM x min, p=0.017), increased OCP (22.6 ± 4.6 Abs-sum vs 20.8 ± 4.1 Abs-sum, p=0.025), shorter APTT (30.9 ± 3.8s vs 33.4 ± 3.6s, p<0.001) and lower antithrombin activity (99, 93-105% vs 104, 100-109%, p<0.05) were observed. Thrombophilia was observed in 22/60 (36%) patients and in 5/63 (7.9%, p<0.001) controls. The only significant difference between thrombophilic and non-thrombophilic patients was higher soluble P-selectin in the former subgroup (22, 20-33 μg/L vs 17, 12-22 μg/L, p=0.012). In women with a history of OC-related VT persistent hypercoagulability was observed, which, however was not augmented by the presence of thrombophilia.

Screening Haemostasis – Looking for Global Assays: The Overall Haemostasis Potential (OHP) Method – A Possible Tool for Laboratory Investigation of Global Haemostasis in Both Hypo- and Hypercoagulable Conditions

A broad spectrum of global haemostatic assays has recently been developed and modified in an attempt to overcome the drawbacks of classical screening tests used for evaluation of coagulation and fibrinolysis. The Overall Haemostasis Potential (OHP) assay is one of such assays. The assay is based on repeated spectrophotometric registration of fibrin-aggregation in citrated plasma, to which small amounts of exogenous thrombin, tissue type plasminogen activator and calcium chloride have been added. The area under the fibrin aggregation curve which then develops is calculated and is the laboratory parameter used for OHP determination. The Overall Coagulation Potential (OCP) and Overall Fibrinolytic Potential (OFP) are supplementary parameters of OHP, providing details of underlying changes in coagulation and/or fibrinolysis. The sensitivity of the assay for detecting hypercoagulation in normal pregnancy, in preeclampsia, some thrombophilias, coronary heart disease, diabetes, stroke and vascular surgery has been evaluated. Since the assay can monitor haemostasis balance in the sample, it may serve as a laboratory tool to determine hypocoagulation, especially in patients with haemophilia A or B. Preliminary findings also indicate that the OHP assay may be useful in the monitoring of anticoagulant treatments. Larger controlled clinical studies are, however, mandatory before a definite conclusion about the usefulness of the method can be drawn.

Thrombin activatable fibrinolysis inhibitor and its relationship to fibrinolysis and inflammation during the acute and convalescent phase of ischemic stroke

To investigate thrombin activatable fibrinolysis inhibitor (TAFI) in ischemic stroke and its relationship to fibrinolysis and inflammation, we investigated 32 patients with ischemic stroke during the acute phase and after 60 days. TAFI antigen levels, global markers of hemostasis (coagulation and fibrinolysis) and inflammatory markers were measured in plasma. TAFI antigen levels were significantly elevated at admission (128%; 109-151%) and at day 1 (129%; 109-152%) compared with day 60 (108%; 91-127%; both P < 0.01) and with healthy control individuals (99%; 76-122%; P < 0.05). In parallel, fibrinolysis assessed as the overall fibrinolysis potential (OFP), part of the overall hemostatic potential assay (OHP), was decreased at all time points compared with control individuals (P < 0.01 for all) and was found to be inversely related to TAFI (r = -0.40; P = 0.0008; n = 20). The OFP and the overall coagulation potential (another part of the OHP assay), and to a lesser degree TAFI, showed significant relationships to C-reactive protein and fibrinogen. In conclusion, elevated TAFI antigen levels may be a consequence of an acute phase reaction, and together with a depressed OFP suggest impaired fibrinolysis in patients with acute ischemic stroke. The OHP method may be useful as a complement to standard hemostatic variables in evaluating hemostasis in stroke patients.

The effect of platelets on fibrin gel structure formed in the presence of recombinant factor VIIa in hemophilia plasma and in plasma from a patient with Glanzmann thrombasthenia

Fibrin gel structure has been shown to be dependent on the thrombin concentration as well as the rate of thrombin generation. Accordingly, factor VIII (FVIII)- and FIX-deficient plasma (hemophilia A and B) form loose fibrin clots with high permeability constants. By adding rFVIIa in vitro to FVIII-deficient plasma containing platelets (frozen and thawed), the fibrin gel permeability constant normalized, indicating that extra rFVIIa (1.2 microg mL(-1) or higher) induced a tight fibrin structure. Thrombin generation is highly dependent on the number of platelets, and in this study it was demonstrated that the addition of rFVIIa (5 microg mL(-1)) normalizes the fibrin gel permeability in samples containing platelets (frozen-thawed) in numbers of at least down to 20 x 10(6) mL(-1). The effect of rFVIIa was not observed when unfrozen platelets instead of frozen-thawed platelets were added. Neither was any effect on the fibrin permeability seen, in the presence of annexin V, known to block the effect of phospholipids on the platelet surface. This indicates an important role of platelet phospholipids for the effect of rFVIIa. A similar effect on the fibrin permeability of rFVIIa was observed when added to platelet-rich plasma from a patient with Glanzmann thrombasthenia. Recombinant FVIIa has been found to induce hemostasis in patients with hemophilia and inhibitors against FVIII/FIX as well as in patients with Glanzmann thrombasthenia, indicating the importance of the formation of a tight fibrin gel structure, more resistant against premature proteolysis, for maintaining hemostasis. In conclusion, the addition of rFVIIa (5 microg mL(-1)) also substantially decreased the permeability constant of fibrin gels formed in FVIII-deficient plasma in the presence of low numbers of frozen-thawed platelets (down to 20 x 10(6) mL(-1)). A similar pattern was obtained in plasma from a Glanzmann patient. No effect was found in the presence of unfrozen instead of frozen-thawed platelets. Annexin V blocked any effect of rFVIIa. A normalization of the overall fibrinolysis potential (OFP) during the same condition supports the effect of rFVIIa on the fibrin permeability in the presence of a limited number of platelets.

Overall haemostatic potential can be used for estimation of thrombin-activatable fibrinolysis inhibitor-dependent fibrinolysis in vivo and for possible follow-up of recombinant factor VIIa treatment in patients with inhibitors to factor VIII.

Thrombin generation induced by recombinant factor VIIa (rFVIIa) in patients with haemophilia and/or inhibitors to factor VIII/IX could enhance generation of thrombin-activatable fibrinolysis inhibitor (TAFI), a recently described link between coagulation and fibrinolysis. TAFI is unstable and it is not easy to measure its active form in vivo. Overall haemostatic potential (OHP) is a novel method for haemostasis estimation, based on determination of the fibrin aggregation curve in which tiny amounts of thrombin are used for activation of clotting. We measured OHP in six patients with inhibitors to factor VIII before injection of rFVIIa and 10 and 120 min thereafter. Overall fibrinolytic potential (OFP) and clot lysis time (CLT) analysed by this method could be used for indirect estimation of TAFI generation. We found no change in pro-TAFI and total TAFI antigen before and after treatment with rFVIIa. OHP was almost undetectable before treatment but increased into the range of normal pooled plasma 10 and 120 min after rFVIIa treatment, as did CLT. However, after addition of potato tuber carboxypeptidase inhibitor, a specific inhibitor of TAFI, the shortening of CLT was lower than that in NPP. OFP was increased in patient plasma both 10 and 120 min after treatment compared with NPP. There was a strong positive correlation between pro-TAFI concentration and shortening of CLT after PTCI addition and a negative correlation between pro-TAFI concentration and OFP 10 min after rFVIIa injection. Thus, rFVIIa normalizes OHP and CLT 10 min after injection. While this improvement slightly decreases, but still exists after 2 hours, it suggests efficacy in bleeding prevention using a protocol based on rFVIIa administration every 2 hours.

Does Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Contribute to Impairment of Fibrinolysis in Patients with Preeclampsia and/or Intrauterine Fetal Growth Retardation?

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a relatively recently described glycoprotein (MM 55 KDa) that can be converted into its active form by the thrombin/thrombomodulin complex and potentially inhibits fibrinolysis. Since it represents a link between coagulation and fibrinolysis, TAFI can be expected to play a part in various clinical conditions associated with a thrombotic tendency. Preeclampsia (PE) and intrauterine fetal growth retardation (IUFGR) are fairly common complications of pregnancy that are characterized by hemostatic abnormalities. TAFI antigen and its influence on hemostasis was investigated in 46 women with PE and/or IUFGR and in 16 normal pregnancies. We found a significant decrease of TAFI antigen in the patient group. Using the recently described method Overall Hemostatic Potential (OHP) in plasma we measured clot lysis time (CLT) and overall fibrinolytic potential (OFP). We found that CLT is prolonged and OFP decreased in patients with PE and/or IUFGR. Since OFP did not increase after addition of the specific inhibitor of TAFI (potato tuber carboxypeptidase inhibitor), it seems that TAFI does not contribute to the impairment of fibrinolysis in these patients. Since serum albumin was decreased together with presence of proteinuria and aminotransferases were increased in the patients, it seems that one explanation for the decrease in TAFI could be reduced hepatic synthesis and an increased loss in urine. It an be speculated that this mechanism can prevent more serious thrombotic complications in patients with PE and/or IUFGR.

A Simple and Rapid Laboratory Method for Determination of Haemostasis Potential in Plasma: II. Modifications for Use in Routine Laboratories and Research Work

To offer a suitable method for use in routine laboratories and research work, some modifications were made in the assay of overall haemostatsis potential (OHP) we developed earlier. Thrombin in a decreased dose with or without tissue-type plasminogen activator was added to plasma for initiation of fibrinogen clotting. Areas under two fibrin-aggregation curves i.e., abovementioned OHP and overall coagulation potential (OCP) were thus created. A difference between the two parameters reflects the overall fibrinolysis potential (OFP), calculated by ((OCP−OHP)/OCP)×100%. To obtain reference ranges, investigations were performed in 142 healthy adults of different ages and in 29 healthy women with a normal pregnancy. In 16 patients suffering from coronary heart disease (CHD), OCPs and OHPs increased but OFPs decreased. In 10 pre-eclamptic women with moderate enhancement of OCP, the OHPs became noticeably higher in most while the OFPs lower. Extremely low or undetectable levels of OHP and OCP were shown in samples of Factors VIII-, IX-, VII-, V-, X- or II-deficient plasma. In 13 healthy volunteers treated with acetylsalicylic acid (ASA), OHPs expectedly declined during the administration and rose again after withdrawal. The above findings demonstrate that the modifications in the present study have rendered the method more effective for detecting haemostatic changes and relevant for monitoring treatments.