Kenny Hansson

When investigating coagulation assays to measure the effect of infused prothrombin (FII) in in vivo coagulopathy models, we found that addition of FII, plasma-derived human FII (pd-hFII) or recombinant human FII (r-hFII), to normal plasma resulted in a concentration-dependent increase in prothrombin time (PT) initiated with Innovin(®) . The effect on PT by addition to plasma of either pd-hFII or r-hFII, using different commercial PT reagents, was studied both by turbidimetry and viscometry. Addition of FII to plasma resulted in increased PT when initiated with Innovin(®) : PT increased with 20% by doubling the concentration. The prolongation of PT became more pronounced with 2-6000 times diluted Innovin(®) . However, by adjustment of the final free Ca(2+) concentration in the assay with diluted Innovin(®) from 8.3 to 1.3 mmol/L, no FII-dependent increase in PT was found. In contrast, no prolongation of PT was found with other commercial PT reagents. A KM = 3 nmol/L was obtained with...

Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. In the present work the specificity of CTI for factor (F) XIIa is questioned. In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 μm CTI (assay concentration 2.4 μm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 μm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF conce...

In the activated partial thromboplastin time (APTT) assay, a variety of nonphysiological reagents is used to induce contact activation. The sensitivity of the APTT response for different thrombin inhibitors has previously been found to be dependent on the used reagent. Recently, infusion of prothrombin (FII) has been used in in-vivo coagulopathy models and its effect has been analyzed in different assays. Therefore, we investigated whether the FII plasma concentration might affect APTT using different commercial reagents, applying both turbidimetry and viscometry. We compared both plasma-derived human FII (pd-hFII) and recombinant human FII (r-hFII). Similar results were found for pd-hFII and r-hFII with different APTT reagents. As expected, no effect on APTT was found by increasing the plasma concentration of FII using APTT reagents consisting of ellagic acid (Actin FS or Actin). Although with Pathromtin SL, consisting of SiO2, only a slight increase was found, with most other comm...

A design strategy was used to identify inhibitors of activated protein C with selectivity over thrombin featured by a basic and/or aromatic functionality for binding to the S2 pocket. Our strongest inhibitor showed an IC50-material value and selectivity for APC vs thrombin similar to a compound previously reported in the literature. However, in contrast to the reference compound, our compound showed a retained coagulant effect of thrombin with increasing substrate concentration in a modified Calibrated Automated Thrombogram (CAT) method. This was likely related to our compound being inactive against FVIIa, while the reference compound showed an IC50 of 8.9 μM. Thus, the higher selectivity of our compound against all relevant coagulation factors likely explained its higher therapeutic potential in comparison to the reference compound. The data indicate that at least a 100-fold selectivity over other serine proteases in the coagulation cascade will be required for an effective APC inhibitor.

Bleeding complications are common in cardiac surgery. Perioperative handling of heparin and protamine may influence the haemostasis. We hypothesized that heparin and protamine dosing based on individual titration curves would improve haemostasis in comparison to standard dosing. Sixty patients scheduled for first time elective coronary artery bypass grafting or valve surgery were included in a prospective randomized study. The patients were randomized to heparin and protamine dosing with Hepcon HMS Plus device or to standard weight and activated clotting time (ACT) based dosing. Blood samples were collected before and 10 minutes, 2 hours and 4 hours after cardiopulmonary bypass. Primary endpoint was endogenous thrombin potential in plasma 2 hours after surgery as assessed by calibrated automated thrombography. Secondary endpoints included total heparin and protamine doses, whole blood clot formation (thromboelastometry) and post-operative bleeding volume and transfusions. Heparin effect was assessed by measuring anti-Xa activity. Endogenous thrombin potential and clot formation deteriorated in both groups after surgery without statistically significant intergroup difference. There were no significant differences between the groups in total heparin and protamine doses, heparin effect, or postoperative bleeding and transfusions at any time point. Significant inverse correlations between anti-Xa activity and endogenous thrombin potential were observed 10 min (r = -0.43, p = 0.001), 2 hours (r = -0.66, p<0.001) and 4 hours after surgery (r = -0.58, p<0.001). In conclusion, the results suggest that perioperative heparin and protamine dosing based on individual titration curves does not improve haemostasis after cardiac surgery. Postoperative thrombin generation capacity correlates to residual heparin effect. www.isrctn.com ISRCTN14201041.

We describe a novel method to immobilize recombinant human tissue factor (rhTF) reconstituted in phospholipid vesicles. The rhTF vesicles were immobilized in a multilayer vesicle structure using cholesterol-DNA tethers spontaneously inserted into the lipid membrane. The properties of the rhTF vesicle surface modification were characterized by surface plasmon resonance biosensor technology. As an application of this surface modification, we investigated its use as a blood coagulation activating surface. The coagulation activating capacity of the surface modification was tested by exposure to human whole blood in a flow chamber. No increase in rhTF levels in the blood was found after passage through the flow chamber, indicating that the rhTF surface modification was stable. Thrombin-antithrombin (TAT) and prothrombin fragment (PF) 1 + 2 levels increased after exposure to the surface, and decreased in a concentration-dependent way upon addition of melagatran (a direct thrombin inhibitor), i.e., coagulation activity triggered by rhTF could be suppressed by anticoagulation. The results with this new thrombogenic surface are promising, and will be further developed into a useful tool for coagulation related investigations, e.g., characterization of anticoagulants and biomaterials.

INTRODUCTION: Thrombin is a key component in the coagulation cascade, and impaired thrombin generation has been linked to increased bleeding after surgical procedures. The aim was to evaluate postoperative thrombin generation capacity in plasma after cardiac surgery, and its potential associations to activity of individual coagulation factors and heparin. MATERIAL AND METHODS: Forty-eight coronary artery bypass grafting patients were included in a prospective observational cohort study. Thrombin generation capacity was analysed in plasma with calibrated automated thrombogram with tissue factor as activator before (baseline), and 2h and 24h after surgery. In addition, plasma activity of coagulation factors II, V, VII, VIII, IX, X, XI, XIII, were determined. Heparin effect was assessed by anti-Xa activity, APTT and thrombin time. RESULTS: Thrombin generation was markedly reduced 2h after surgery compared to baseline. Peak levels decreased with median 74% (interquartile range 52-90), p...