Arne W.J.H. Dielis

Coagulation factors and the protein C system as determinants of thrombin generation in a normal population

Summary.  Background: Thrombin generation is a powerful tool to probe overall plasma coagulability.

Monitoring thrombin generation: Is addition of corn trypsin inhibitor needed?

Thrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the near future. However, robust preanalytical conditions may be required, and one factor that has been reported is in-vitro contact activation that might influence in-vitro measurements of thrombin generation and thereby act as an unpredictable pre-analytical variable. The aim of the current study was to investigate the influence of contact activation and the necessity of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurements at low tissue factor (TF) concentrations. Thrombin generation was performed using the calibrated automated thrombinoscopy (CAT), thereby determining the endogenous thrombin potential (ETP), peak height, and the lag time, in plasma obtained from healthy volunteers. Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.5 pM TF or higher, as demonstrated by unaltered ETP and lag time values between analyses with and without CTI. Addition of CTI before blood collection reduced thrombin generation triggered with 0.5 pM TF: both the ETP and peak height were significantly reduced compared to no CTI addition. In contrast, thrombin generation remained unaltered at a 1 pM TF trigger or above. This study demonstrates that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.5 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring thrombin generation at TF concentrations of >/=1 pM.

Assessment of thrombin generation II: Validation of the Calibrated Automated Thrombogram in platelet-poor plasma in a clinical laboratory

Assessment of thrombin generation II: Validation of the Calibrated Automated Thrombogram in platelet-poor plasma in a clinical laboratory -

Thrombin generation in patients with a first acute myocardial infarction

Summary.  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.

The Prothrombotic Paradox of Hypertension: Role of the Renin-Angiotensin and Kallikrein-Kinin Systems

Despite increased pulsatile stress, thrombotic rather than hemorrhagic events represent a major complication of hypertension. The pathophysiology of thrombosis in hypertension involves the interaction among vascular endothelium and particularly the renin-angiotensin and kallikrein-kinin systems. Because hypertension is often associated with some degree of inflammation, the combination of chronic inflammation and chronic shear stress may convert the normal anticoagulant endothelium into a procoagulant surface, expressing tissue factor. Activation of the renin-angiotensin system leads to activation of nuclear factor kappaB-dependent proinflammatory genes, also accelerating the expression of tissue factor. Renin-angiotensin and kallikrein-kinin systems interact at several levels to modulate coagulation, fibrinolysis, and vasodilatation in such a way that these 2 systems could have a major influence on the occurrence of thrombotic complications. Treatment with angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists may favorably influence the balance between the renin-angiotensin and kallikrein-kinin axis, regulating blood pressure as well as reducing the risk of thrombosis, which may explain part of the clinical efficacy of these drugs.

Thrombin generation in patients after acute deep-vein thrombosis

Thrombin generation measurement may be of value for assessing the risk of venous thromboembolism, but its long term profile has not been assessed in patients. We evaluated thrombin generation by Calibrated Automated Thrombogram (CAT) in plasma during follow up of 104 consecutive patients after an acute episode of deep venous thrombosis. Blood was drawn three times over the course of 24 months. Thrombin generation was measured in absence and presence of thrombomodulin and compared to a reference range derived from thrombin generation curves in 137 healthy volunteers. Thrombin generation of patients showed significantly higher endogenous thrombin potential (ETP) and peak height compared to the reference population. Differences were more pronounced in assays triggered with 1 pM TF. Inhibition by thrombomodulin was attenuated in patients off anticoagulants as compared to the reference population (21% vs. 42.2%, p < 0.0001); inhibition in patients on anticoagulant treatment was less pronounced (9.7%, p < 0.0001). Protein C activity, protein S antigen as well as free protein S showed highly negative correlation with ETP in all patients. A significant negative relation was found between FVIII levels and thrombomodulin induced reduction of ETP and peak height. In conclusion, thrombin generation by CAT reflects changes in coagulation status in patients following a thromboembolic event and is most sensitive at CAT analysis triggered with 1 pM TF. A role for factor VIII as an important attributable cause of hypercoagulability is reflected by the reduced inhibitory effect of thrombomodulin at high factor VIII levels.

Changes in fibrinolytic activity after angiotensin II receptor blockade in therapy‐resistant hypertensive patients

Summary.  Background: In hypertensive patients, the activated renin–angiotensin system induces a prothrombotic state resulting from imbalance between coagulation and fibrinolysis. Although blood pressure cannot be regulated in therapy‐resistant hypertensive patients, they may still be responsive to medication that attenuates the renin–angiotensin system. Objective: Our objective was to study possible attenuating properties of angiotensin II type 1 receptor blockers (AT1RBs) on the prothrombotic state in therapy‐resistant hypertensive patients, focusing on parameters of fibrinolysis and coagulation. Methods: Fourteen therapy‐resistant hypertensive patients received AT1RB eprosartan infusion (45 and 150 μg kg–1) (study group), and 33 therapy‐resistant hypertensive patients received saline (0.9%) infusion (control group) prior to renal angiography. Baseline values of parameters of coagulation and fibrinolysis were set at 1.00, and relative changes were calculated. Results: Plasminogen activator inhibitor type 1 (PAI‐1) antigen showed non‐significant decreases in both the study group (arterial 1.00–0.45, venous 1.00–0.42) and control group (arterial 1.00–0.84, venous 1.00–0.88). PAI‐1 activity significantly decreased in the study group (arterial 1.00–0.72, venous 1.00–0.71) and control group (arterial 1.00–0.83, venous 1.00–0.94). In the study group, tissue‐type plasminogen activator (t‐PA) antigen decreased significantly (arterial 1.00–0.62, venous 1.00–0.67), whereas t‐PA activity significantly increased (arterial 1.00–6.15, venous 1.00–2.66). In the control group, t‐PA antigen remained unchanged. No changes were observed in blood pressure during and after infusion of eprosartan. Conclusion: Therapy‐resistant hypertensive patients show beneficial changes in fibrinolytic activity after infusion of a non‐pressor dose of AT1RB.

Thrombin Generation in the Glasgow Myocardial Infarction Study

Background Thrombin is a key protease in coagulation also implicated in complex pathology including atherosclerosis. To address the role of thrombin in relation to myocardial infarction (MI) we explored thrombin generation analysis in plasma from patients and controls that had participated in the Glasgow MI Study (GLAMIS). Methods Thrombin generation at 1 and 2 pM TF and with and without thrombomodulin (TM) was performed on plasmas from 356 subjects (171 cases, 185 age and sex matched controls) from GLAMIS collected between 3 and 9 months after the MI event. Results Although thrombin generation was slightly delayed in cases (lag time increased from 3.3 to 3.6 min) at the highest trigger, the overall potential to generate thrombin was increased by 7% for the ETP and by 15% for the peak height (both at the 1 pM TF trigger) in cases. Addition of TM did not reveal differences. Furthermore, an increased thrombin generation was associated with MI [normalized ETP: adjusted OR for the highest percentile = 2.4 (95% CI 1.3–4.5) and normalized peak height: adjusted OR = 2.6 (1.3–5.0)] at the lowest trigger; normalized ETP and peak height being 2.1 (1.1–3.8) and 2.0 (1.0–4.1) at the higher 2 pM trigger. Conclusion In GLAMIS, patients with a previous MI had an increased thrombin generation compared to controls. The absence of a clear difference in TM reduction suggests an unaltered anticoagulant activity in these patients. Further research is needed in order to unravel the underlying mechanisms of enhanced thrombin generation after MI.

Thrombomodulin-modified thrombin generation after in vivo recombinant factor VIII treatment in severe hemophilia A

Thrombin generation has been shown to reflect coagulation potential and factor VIII (FVIII) levels in patients with hemophilia A. This study shows that thrombin generation in the presence of thrombomodulin reflects plasma FVIII levels better. Background Thrombin generation has been shown to reflect coagulation potential and factor VIII (FVIII) levels in patients with hemophilia A. We hypothesize that thrombin generation in the presence of thrombomodulin reflects plasma FVIII levels better. Design and Methods Plasma FVIII levels were determined chromogenically and thrombin generation was measured with and without thrombomodulin in 12 patients with severe hemophilia A. Blood was sampled at baseline and 15 min, 1, 3, 6, 24 and 48 hours after recombinant FVIII administration. Results FVIII administration restored the decreased baseline thrombin generation (reflected by endogenous thrombin potential, peak height, slope and time to peak). Lag time did not change. All thrombin generation parameters except time to peak returned to baseline within 48 hours, while plasma FVIII concentration was increased and time to peak shortened. Endogenous thrombin potential and peak height showed wide inter-individual variation, with strong intra-individual correlations. Addition of thrombomodulin to the assay shortened time to peak and decreased endogenous thrombin potential and peak height. The decrease in peak height was almost completely offset by FVIII administration. Multiple linear regression analysis revealed thrombomodulin-modified thrombin generation to be a moderately better predictor of plasma FVIII levels than thrombin generation in the absence of thrombomodulin (adjusted R2 0.79 vs. 0.71). Conclusions Addition of thrombomodulin has pronounced effects on all parameters of thrombin generation. This thrombomodulin-modified thrombin generation assay better reflects plasma FVIII levels than thrombin generation in the absence of thrombomodulin.