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.

Thrombin Generation as a Method to Identify the Risk of Bleeding in High Clinical-Risk Patients Using Dual Antiplatelet Therapy.

Background: Patients using dual antiplatelet therapy after percutaneous coronary intervention are at risk for bleeding. It is currently unknown whether thrombin generation can be used to identify patients receiving dual antiplatelet therapy with increased bleeding risk. Objectives: To investigate whether thrombin generation measurement in plasma provides additional insight into the assessment of bleeding risk for high clinical-risk patients using dual antiplatelet therapy. Methods: Coagulation factors and thrombin generation in platelet-poor plasma were measured in 93 high clinical-risk frail patients using dual antiplatelet therapy after percutaneous coronary intervention. During 12-month follow-up, clinically relevant bleedings were reported. Thrombin generation at 1 and 6 months after percutaneous coronary intervention was compared between patients with and without bleeding events. Results: One month after percutaneous coronary intervention, the parameters of thrombin generation, endogenous thrombin potential, peak height, and velocity index were significantly lower in patients with bleeding in the following months compared to patients without bleeding. At 6 months follow-up, endogenous thrombin potential, peak height, and velocity index were still (significantly) decreased in the bleeding group as compared to non-bleeders. Thrombin generation in the patients' plasma was strongly dependent on factor II, V, and VIII activity and fibrinogen. Conclusion: High clinical-risk patients using dual antiplatelet therapy with clinically relevant bleeding during follow-up show reduced and delayed thrombin generation in platelet-poor plasma, possibly due to variation in coagulation factors. Thus, impaired thrombin-generating potential may be a "second hit" on top of dual antiplatelet therapy, increasing the bleeding risk in high clinical-risk patients. Thrombin generation has the potential to improve the identification of patients using dual antiplatelet therapy at increased risk of bleeding.

Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline.

Light transmission aggregometry (LTA) is considered the gold standard method for evaluation of platelet function. However, there are a lot of variation in protocols (pre-analytical procedures and agonist concentrations) and results. The aim of our study was to establish a national LTA protocol, to investigate the effect of standardization and to define national reference values for LTA. The SSC guideline was used as base for a national procedure. Almost all recommendations of the SSC were followed e.g. no adjustment of PRP, citrate concentration of 109 mM, 21 needle gauge, fasting, resting time for whole blood and PRP, centrifugation time, speed and agonists concentrations. LTA of healthy volunteers was measured in a total of 16 hospitals with 5 hospitals before and after standardization. Results of more than 120 healthy volunteers (maximum aggregation %) were collected, with participating laboratories using 4 different analyzers with different reagents. Use of low agonist concentrations showed high variation before and after standardization, with the exception of collagen. For most high agonist concentrations (ADP, collagen, ristocetin, epinephrine and arachidonic acid) variability in healthy subjects decreased after standardization. We can conclude that a standardized Dutch protocol for LTA, based on the SSC guideline, does not result in smaller variability in healthy volunteers for all agonist concentrations.

Vascular protective effect of aspirin and rivaroxaban upon endothelial denudation of the mouse carotid artery.

While in recent trials the dual pathway inhibition with aspirin plus rivaroxaban has shown to be efficacious in patients with atherosclerotic cardiovascular disease, little is known about the effects of this combination treatment on thrombus formation and vascular remodelling upon vascular damage. The aim of this study was to examine the effects of aspirin and/or rivaroxaban on injury-induced murine arterial thrombus formation in vivo and in vitro, vessel-wall remodelling, and platelet-leukocyte aggregates. Temporary ligation of the carotid artery of C57BL/6 mice, fed a western type diet, led to endothelial denudation and sub-occlusive thrombus formation. At the site of ligation, the vessel wall stiffened and the intima-media thickened. Aspirin treatment antagonized vascular stiffening and rivaroxaban treatment led to a positive trend towards reduced stiffening. Local intima-media thickening was antagonized by both aspirin or rivaroxaban treatment. Platelet-leukocyte aggregates and the number of platelets per leukocyte were reduced in aspirin and/or rivaroxaban treatment groups. Furthermore, rivaroxaban restricted thrombus growth and height in vitro. In sum, this study shows vascular protective effects of aspirin and rivaroxaban, upon vascular injury of the mouse artery.

OC-08 - Multiple functional defects in platelets from thrombocytopenic cancer patients undergoing chemotherapy.

INTRODUCTION: Severe thrombocytopenia (≤50×10(9) platelets/L) is often the consequence of hematological malignancies and intensive chemotherapy. The risk of clinically significant bleeding is increased in these patients, despite the use of prophylactic platelet transfusions. The fact that there is no clear correlation between the platelet count and the risk of hemorrhage, suggests that there are other contributing factors. The contribution of impairments in platelet and coagulant function remains poorly understood. AIM: In patients with chemotherapy-induced thrombocytopenia due to hematological malignancies, we evaluate platelet and coagulant functions and determine the effects of platelet transfusion. Ultimately, we can identify specific hemostatic factors that aid in the prediction of bleeding. MATERIALS AND METHODS: In total 58 patients were included and blood was collected before and, if indicated (≤10×10(9) platelets/L), 1 hour after transfusion with platelet concentrate. Platelet function was assessed using flow cytometry by determining: 1) integrin αIIbß3 activation (PAC-1 antibody), 2) P-selectin expression (anti-P-selectin antibody), 3) phosphatidylserine exposure (Annexin-V) and 4) intracellular calcium (Fluo-4 AM). Factor levels were determined in plasma. Thrombus and fibrin formation was assessed by perfusion of whole blood over a collagen-tissue factor surface at a shear rate of 1,000 s-1. RESULTS: Platelets from the thrombocytopenic patients before transfusion showed markedly reduced integrin αIIbß3 activation and P-selectin expression in response to thrombin, collagen-related peptide and ADP, compared to healthy donor platelets. Also, agonist-induced intracellular calcium fluxes were greatly reduced. However, calcium fluxes with thapsigargin, a SERCA pump inhibitor, were similar in patient and control platelets, suggesting a normal calcium store content in the patient platelets. Furthermore, phosphatidylserine exposure was increased in unstimulated patient platelets compared to control platelets (8.2 vs. 1.8%, p<0.0001). Coagulation factor levels were within the normal range, with the exception of von Willebrand factor and fibrinogen levels, which were elevated. Platelet transfusion partly recovered the platelet integrin αIIbß3 activation and P-selectin expression induced by agonists. Platelet deposition (6.7 vs. 1.7%, p<0.0001) and fibrin formation (7.6 vs. 0.9%, p=0.0005) under flow conditions were substantially improved after platelet transfusion. CONCLUSIONS: Platelets from cancer patients undergoing chemotherapy appear to display impaired functional responses to activating stimuli. Platelet transfusion partly restores these functional defects, resulting in improved thrombus and fibrin formation.

Increased coagulation and fibrinolytic potential of solvent-detergent plasma: a comparative study between Omniplasma and fresh frozen plasma.

BACKGROUND AND OBJECTIVES: In this study, differences in levels of proteins involved in coagulation and fibrinolysis were compared between fresh frozen (quarantine plasma) and Omniplasma. Furthermore, thawing conditions and plasma stability after thawing were studied. MATERIALS AND METHODS: 10 Omniplasma and 10 quarantine plasma units were used to study different procoagulation, anticoagulation and fibrinolytic parameters. Analysis took place at different time-points during plasma storage at 2-6°C. RESULTS: At baseline, significant reduced levels of factor V, free protein S, α2-antiplasmin and tPA-induced ROTEM lysis time were observed in Omniplasma as compared to quarantine plasma. Moreover, thrombin generation, IXa-AT complex levels and factor XIa were significantly increased in Omniplasma. The majority of the parameters studied remained stable in Omniplasma 48 h after thawing, with the exception of factor VIII (decrease) and IXa-AT (increase). CONCLUSION: Our results suggest an increased coagulation potential, presumingly as a result of contact activation during the production process and also, an increased fibrinolytic potential in Omniplasma. The stability of Omniplasma, based upon the different parameters studied, is comparable to Q-plasma. A maximum post-thawing time of 48 hfor Omniplasma can be suggested.

Biomarkers for Prediction of Central Venous Catheter Related-Thrombosis in Patients With Hematological Malignancies.

OBJECTIVE: In a prospective setting, we aimed to find associations between biomarkers of the hemostatic system and the occurrence of central venous catheter (CVC)-related thrombosis in patients with hematological malignancies undergoing intensive chemotherapy. METHODS: The study was conducted between July 2006 and August 2010 at the University Hospital Maastricht, the Netherlands. Consecutive adult patients with hematological malignancies who were going to receive a CVC for intensive chemotherapy were included. The primary end points were (a) symptomatic CVC-related thrombosis and (b) CVC-related infections. Blood samples were taken directly after catheterization, and easy to determine biomarkers (platelet count, leukocyte count, and hemoglobin level) in combination with blood group, factor VIII (FVIII), plasminogen activator inhibitor 1 (PAI-1), activated protein C (APC) resistance, and free protein S antigen were determined. RESULTS: Blood was collected and analyzed from 168 patients. The incidence of symptomatic CVC-related thrombosis was 9%. In univariate analysis, white blood cell count >10.6 × 109/L, mean FVIII activity, and PAI-1 >12.2 IU/mL were found to be associated with the development of symptomatic CVC-related thrombosis. CONCLUSION: Elevated leukocyte count, high PAI-1, and high FVIII were associated with an increased incidence of symptomatic CVC-related thrombosis. We hope in future that simple, easy to determine laboratory tests that reflect the hemostatic and fibrinolytic activity in combination with clinical parameters may help to identify hematological patients at highest risk of CVC-related thrombosis and help to tailor the management of thromboprophylaxis in hematological patients undergoing CVC placement.

The hypercoagulable profile of patients with stent thrombosis.

OBJECTIVE: Coronary stent thrombosis is a devastating complication after percutaneous coronary intervention (PCI). The mechanisms underlying stent thrombosis are multifactorial. Whether the coagulation system is involved in the pathophysiology of stent thrombosis is unclear. We hypothesised that thrombin generation, reflecting the coagulation potential, is enhanced in patients with stent thrombosis. METHODS: A case-control study was performed, including 63 patients with PCI: 23 cases (stent thrombosis) and 40 controls (no stent thrombosis). Thrombin generation was measured using 0, 1 and 5 pM tissue factor (TF) triggers. Active site-inhibited factor VIIa (ASIS) and recombinant thrombomodulin were added to study the contact activation system and the protein C pathway, respectively. RESULTS: Thrombin generation was significantly increased for all TF triggers in cases compared with controls. Addition of ASIS to the measurement without exogenous TF revealed significantly enhanced contact activation in cases compared with controls; mean peak height: 241 vs 183 nM. Thrombin generation was also significantly increased in cases compared with controls in the presence of exogenous TF; mean peak height: 263 vs 233 nM (5 pM TF). Addition of thrombomodulin reduced thrombin generation by 23% in cases and 31% in controls (p<0.018), suggesting alterations in the protein C pathway in cases. CONCLUSIONS: This is the first study that suggests the involvement of the coagulation system in stent thrombosis. Stent thrombosis patients showed a hypercoagulable state, most likely caused by enhanced contact activation and attenuation of anticoagulation by the protein C pathway.

Desmopressin treatment improves platelet function under flow in patients with postoperative bleeding.

BACKGROUND: Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. OBJECTIVES: To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. PATIENTS/METHODS: Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3 µg kg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. RESULTS: Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factor VIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116 IU mL(-1) (interquartile range [IQR] 102-154 IU mL(-1) ) to a median of 160 IU mL(-1) (IQR 126-187 IU mL(-1) ) (P = 0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. In vitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. CONCLUSIONS: Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.

Increased factor XIa levels in patients with a first acute myocardial infarction: the introduction of a new thrombin generation based factor XIa assay.

OBJECTIVE: One of the contributing mechanisms in acute myocardial infarction (AMI) is plasma hypercoagulability. Recently, it was suggested that factor XI activation might play a role in atherothrombosis. To quantify factor XIa plasma levels, we developed a new thrombin generation based assay and hypothesized that in AMI patients factor XIa levels are increased during the acute thrombotic event. METHODS: A prospective cohort study was performed including 56 patients with first AMI. Blood was collected upon admission and after 6 months. Reference blood samples were obtained from 30 apparently healthy control subjects. Plasma samples were diluted (1:5) in factor XI deficient plasma and factor XIa plasma levels were established using a reference curve (0-12.5 pM factor XIa) and an inhibitory anti-factor XIa antibody. The established FXIa concentrations were related to the 1-year outcome. RESULTS: Factor XIa plasma concentrations were significantly increased in AMI patients on admission compared to 6 months after the event (3.7 pM [2.7-5.5] vs. 2.8 [1.9-4.3], median ± IQR; P=0.001) and compared to healthy controls (3.7 pM [2.7-5.5] vs. 2.7 [1.6-4.2], median ± IQR; P=0.004). However, a high factor FXIa level at baseline was not significantly associated with a recurrent cardiovascular event (OR 1.26, 95%CI 0.33-4.7). CONCLUSIONS: This study presents the first application of a new thrombin generation based factor XIa assay, showing significantly increased factor XIa levels in AMI patients on admission compared to 6 months after the event and compared to healthy controls. The factor XIa concentration was not associated with the risk of recurrence.

In vivo effects of foam sclerotherapy on coagulation.

OBJECTIVE: The aim of this study was to evaluate whether foam sclerotherapy (FS) induces changes in CAT (calibrated automated thrombinography) and other coagulation parameters which could indicate an increased risk of thrombotic events. METHODS: Blood samples from eight patients treated with FS were taken before treatment and 30 minutes, one and four hours and one week after treatment. CAT parameters (ETP1n, Peak1n, Lag time 1), thrombin antithrombin complexes (TAT), d-dimers, fibrinogen, Von Willebrand (vWf Ag) factor and platelet-derived microparticles (MIPAs) were measured. RESULTS: Significant changes over time for Peak1n, fibrinogen, d-dimers, vWfAg and TAT complexes were observed. CAT parameters decreased over time, except for Lag time 1. D-dimers and TAT complexes increased and fibrinogen, vWf Ag, MIPA's decreased during the first hours. CONCLUSION: The findings in this study support the hypothesis that FS initiate coagulation pathways, but there is no evidence that this activation results in an increased thrombosis risk.

Development of novel membranes for blood purification therapies based on copolymers of N-vinylpyrrolidone and n-butylmethacrylate.

Developments in membrane based blood purification therapies often come with longer treatment times and therefore longer blood-material contact, which requires long-term membrane biocompatibility. In this study, we develop for the first time membranes for blood purification using the material SlipSkin™, which is a copolymer, made from N-vinylpyrrolidone (NVP) and butylmethacrylate (BMA). Specific attention is focused on understanding the mechanism of pore formation and the tailoring of the membrane mechanical and transport properties to obtain the optimal membrane for blood purification therapies. Polymer composition, solvent type and solvent evaporation time influence membrane morphology and membranes with sieving properties of cascade filters in plasma fractionation applications are developed. The new membranes have very good blood compatibility properties; in fact compared to benchmark flat membranes currently used in the clinic, they have lower platelet adhesion while all other properties (contact activation, thrombogenicity, leukocyte adhesion, hemolysis and complement activation) are also very good and comparable to the benchmarks.

Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method.

BACKGROUND: Thrombin generation assays are sensitive methods for assessment of the overall clotting potential of plasma, but, despite their common use in thrombosis research, standardization of preanalytic conditions is lacking. In order to set up a standardized protocol, we analyzed different preanalytic variables and validated the calibrated automated thrombogram method. METHODS AND RESULTS: Thrombin generation was assessed with 0, 1 and 5 pm tissue factor (TF). Variations in thrombin generation were mostly attributable to the type of collection tube, mainly because of variations in contact activation. The collection tube also determined the influence of other preanalytic variables on thrombin generation, e.g. the need for a discard tube, the storage of whole blood, and the centrifugation method. Regarding the collection system, blood drawn through intravenous catheters or butterfly needles showed significantly more hemolysis than blood obtained with venipuncture using conventional needles. The results showed that a discard tube is still needed for thrombin generation measurements. After blood collection, whole blood is best centrifuged immediately, to prevent activation or degradation of coagulation proteins, and a second centrifugation step at 10,000 × g is recommended. After thawing, plasma is best analyzed immediately, as storage resulted in thrombin generation results outside the 10% range of the reference sample. On the basis of these results, we set up an in-house standardized protocol, which was used for validation, resulting in coefficients of variations of < 15% for all derived parameters with both the 1 and 5 pm TF triggers. CONCLUSION: Thrombin generation was greatly influenced by preanalytic conditions, demonstrating the need for an international standardized protocol.

Platelet- and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa.

BACKGROUND: The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. OBJECTIVES: To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. METHODS: Microparticles were isolated from calcium ionophore-stimulated platelets, erythrocytes and monocytic THP-1 cells. MPs were quantified, characterized for cell-specific antigens and analyzed for TF, PS exposure and their thrombin-generating potential. RESULTS: The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet- and erythrocyte-derived MPs (< 1 fM nM(-1) PS), whereas monocyte-derived MPs exposed TF (32 fM nM(-1) PS). Platelet-, erythrocyte- and monocyte-derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet- and erythrocyte-derived MPs, but interfered with monocyte MP-triggered coagulation. Platelet- and erythrocyte-derived MPs completely failed to induce thrombin generation in FXII-deficient plasma. In contrast, monocyte-derived MPs induced similar thrombin generation in normal vs. FXII-deficient plasma. CONCLUSION: MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII-dependent manner. In contrast, monocyte-derived MPs trigger coagulation predominantly via TF.

Platelet concentrate transport in pneumatic tube systems--does it work?

Blood sample transport via pneumatic tube systems (PTS) reduces the turnaround time of laboratories, but it might influence analysis results. Its effect on platelet concentrates (PCs) is not known. Platelet function was investigated after single and multiple PTS transport in comparison with storage and irradiation. Optical and impedance aggregation, CD-62, and microparticles changed as a result of storage, but not due to transport. Irradiation lowered platelet function independently. Multiple transport impaired thrombin receptor-activating peptide-induced aggregation. This investigation demonstrates the feasibility of PTS transport. As platelet function depends on storage, it may be more important to transfuse fresh PCs.

ATP-gated P2X1 ion channels protect against endotoxemia by dampening neutrophil activation.

BACKGROUND: In sepsis, extracellular ATP, secreted by activated platelets and leukocytes, may contribute to the crosstalk between hemostasis and inflammation. Previously, we showed that, in addition to their role in platelet activation, ATP-gated P2X(1) ion channels are involved in promoting neutrophil chemotaxis. OBJECTIVES: To elucidate the contribution of P2X(1) ion channels to sepsis and the associated disturbance of hemostasis. METHODS: We used P2X(1) (-/-) mice in a model of lipopolysaccharide (LPS)-induced sepsis. Hemostasis and inflammation parameters were analyzed together with outcome. Mechanisms were further studied ex vivo with mouse and human blood or isolated neutrophils and monocytes. RESULTS: P2X(1) (-/-) mice were more susceptible to LPS-induced shock than wild-type mice, despite normal cytokine production. Plasma levels of thrombin-antithrombin complexes were higher, thrombocytopenia was worsened, and whole blood coagulation time was markedly reduced, pointing to aggravated hemostasis disturbance in the absence of P2X(1). However, whole blood platelet aggregation occurred normally, and P2X(1) (-/-) macrophages displayed normal levels of total tissue factor activity. We found that P2X(1) (-/-) neutrophils produced higher amounts of reactive oxygen species. Increased amounts of myeloperoxidase were released in the blood of LPS-treated P2X(1) (-/-) mice, and circulating neutrophils and monocytes expressed higher levels of CD11b. Neutrophil accumulation in the lungs was also significantly augmented, as was lipid peroxidation in the liver. Desensitization of P2X(1) ion channels led to increased activation of human neutrophils and enhanced formation of platelet-leukocyte aggregates. CONCLUSIONS: P2X(1) ion channels play a protective role in endotoxemia by negatively regulating systemic neutrophil activation, thereby limiting the oxidative response, coagulation, and organ damage.

Thrombin generation assay using factor IXa as a trigger to quantify accurately factor VIII levels in haemophilia A.

BACKGROUND: The available methods for measuring factor VIII (FVIII) activity suffer reportedly from lack of sensitivity and precision in the < 1 IU dL(-1) range. This precludes correlation of clinical phenotype with FVIII levels. OBJECTIVES: To study a possible association between clinical phenotype in patients with FVIII levels < 1 IU dL(-1). METHODS/RESULTS: The FIXa-driven FVIII assay (FVIII-CAT) has a detection limit of 0.05 IU dL(-1). For the range of 0-2 IU dL(-1) FVIII, the intra-assay coefficient of variation (CV) is around 2% and the inter-assay CV is about 8%. We tested 30 hemophiliacs with FVIII:C between < 1 and 6 IU dL(-1) as measured in the one-stage clotting assay using the FVIII-CAT assay. For genetic defects related to moderate hemophilia, the FVIII-CAT test finds FVIII levels that are in good agreement with those determined with the one-stage assay. Of the 21 hemophilic patients with FVIII < 1 IU dL(-1), four patients exhibited a mild bleeding phenotype. When we applied TF-initiated thrombin generation, patients with a mild clinical phenotype showed significantly higher endogenous thrombin potentials. CONCLUSION: The novel developed FVIII assay measures accurately FVIII levels below 1 IU dL(-1). Its application demonstrated that the clinical heterogeneity in individuals with < 1 IU dL(-1) FVIII is not associated with their FVIII level.

Factor XII activation is essential to sustain the procoagulant effects of particulate matter.

BACKGROUND: Particulate matter (PM) is a key component of ambient air pollution and has been associated with an increased risk of thrombotic events and mortality. The underlying mechanisms remain unclear. OBJECTIVES: To study the mechanisms of PM-driven procoagulant activity in human plasma and to investigate mainly, the coagulation driven by ultrafine particles (UFPs; < 0.1 µm) in genetically modified mice. METHODS: Thrombin generation in response to PM of different sizes was assessed in normal human platelet-poor plasma, as well as in plasmas deficient in the intrinsic pathway proteases factors XII (FXII) or XI (FXI). In addition, UFPs were intratracheally instilled in wild-type (WT) and FXII-deficient (FXII(-/-) ) mice and plasma thrombin generation was analyzed in plasma from treated mice at 4 and 20 h post-exposure. RESULTS: In normal human plasma, thrombin generation was enhanced in the presence of PM, whereas PM-driven thrombin formation was completely abolished in FXII- and FXI-deficient plasma. UFPs induced a transient increase in tissue factor (TF)-driven thrombin formation at 4 h post-instillation in WT mice compared with saline instillation. Intratracheal instillation of UFPs resulted in a procoagulant response in WT mice plasma at 20 h, whereas it was entirely suppressed in FXII(-/-) mice. CONCLUSIONS: Overall, the data suggest that PM promotes its early procoagulant actions mostly through the TF-driven extrinsic pathway of coagulation, whereas PM-driven long lasting thrombogenic effects are predominantly mediated via formation of activated FXII. Hence, FXII-driven thrombin formation may be relevant to an enhanced thrombotic susceptibility upon chronic exposure to PM in humans.

Thrombin generation in patients with a first acute myocardial infarction.

BACKGROUND: Despite improved treatment options, myocardial infarction is still an important cause of morbidity and mortality. One of the contributing mechanisms in the acute myocardial infarction (AMI) is plasma hypercoagulability. METHODS: We investigated hypercoagulability in 135 (first) patients with AMI using thrombin generation (TG) testing. TG testing was performed in plasmas, drawn upon admission and before medication administration, and subsequently after 4 days, 3 and 6 months. Further, we evaluated determinants of thrombin generation using multiple regression analysis of major coagulation proteins and inhibitors. Admission TG results were also related to 1-year outcome: cardiovascular death, recurrent myocardial infarction, a second coronary intervention [percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG)] and ischemic stroke. RESULTS: At day 0, the TG parameters peak height, endogenous thrombin potential (ETP) and lag time were increased compared with a reference population. Peak height and lag time stayed persistently increased in patients. The lowest half of the ETP values was statistically not significantly associated with an occurrence of endpoints. The lowest half of the ETP values combined with the upper half of the D-dimer values were associated with endpoints; odds ratio 5.8 (1.1-30.7). Tissue factor pathway inhibitor (TFPI) seems to be an important determinant of TG in AMI and healthy persons. CONCLUSIONS: TG reflects acute hypercoagulability during AMI and partly also in the 6-month period after the acute event. TG shows a trend of an inverse association with risk of recurrent ischemic cardiovascular complications. Unraveling mechanisms in TG might improve our understanding of the pathophysiology of AMI and direct future improvements in medical care.