The Clotting Trigger Is an Important Determinant for the Coagulation Pathway In Vivo or In Vitro-Inference from Data Review.

Blood coagulation comprises a series of enzymatic reactions leading to thrombin generation and fibrin formation. This process is commonly illustrated in a waterfall-like manner, referred to as the coagulation cascade. In vivo, this "cascade" is initiated through the tissue factor (TF) pathway, once subendothelial TF is exposed and bound to coagulation factor VII (FVII) in blood. In vitro, a diminutive concentration of recombinant TF (rTF) is used as a clotting trigger in various global hemostasis assays such as the calibrated automated thrombogram, methods that assess fibrin turbidity and fibrin viscoelasticity tests such as rotational thromboelastometry. These assays aim to mimic in vivo global coagulation, and are useful in assessing hyper-/hypocoagulable disorders or monitoring therapies with hemostatic agents. An excess of rTF, a sufficient amount of negatively charged surfaces, various concentrations of exogenous thrombin, recombinant activated FVII, or recombinant activated FIXa are also used to initiate activation of specific sub-processes of the coagulation cascade in vitro. These approaches offer important information on certain specific coagulation pathways, while alterations in pro-/anticoagulants not participating in these pathways remain undetectable by these methods. Reviewing available data, we sought to enhance our knowledge of how choice of clotting trigger affects the outcome of hemostasis assays, and address the call for further investigations on this topic.

A fibrinogen concentrate Haemocomplettan (Riastap) or a Factor XIII concentrate Fibrogammin combined with a mini dose of tranexamic acid can reverse the fibrin instability to fibrinolysis induced by thrombin- or FXa-inhibitor.

To assess whether Haemocomplettan(®) (fibrinogen concentrate) or Fibrogammin(®) (Factor XIII concentrate) can be used to manage bleeding complications of antithrombotic treatment, we examined a normal plasma pool spiked with AR-H067637 (thrombin inhibitor) or rivaroxaban (activated factor X-inhibitor), to which one of the concentrates was added. Fibrin network permeability (Ks), images of Scanning Electron Microscopy (SEM) and Clot Lysis Time (CLT) were examined. Both inhibitors increased the Ks levels, which could be fully or partly reversed by Haemocomplettan(®) or Fibrogammin(®) respectively. However, these modified clots with tightened network remained non-resistant to fibrinolysis, shown as unaffected CLT. Tranexamic acid at a very low concentration (0·4 mg/ml) aided the two concentrates to stabilize the clots, where the prolongation of CLT was more pronounced for a lower dose than a higher dose of Haemocomplettan(®) while Fibrogammin(®) brought the greatest delay to CLT out of all additions. These observations were partly supported by SEM images, displaying alterations of fibrin fibre arrangement known to influence fibirinolysis. The in vitro data suggest that Haemocomplettan(®) or Fibrogammin(®) given in combination with a mini dose of tranexamic acid may slow down the natural clearance of fibrin clot by plasmin and thus prevent patients from haemorrhagic complications during antithrombotic therapy.

Modifications of flow measurement to determine fibrin gel permeability and the preliminary use in research and clinical materials.

Our earlier investigations employing a flow measurement have yielded intriguing findings as to what governs the fibrin network porosity. To make the method suitable for use by more groups with various laboratory conditions, sample materials or study purposes, we simplified the essential equipment and thereby minimized the sample volume to 250 microl in comparison with the need for 3000 microl in the previous method. To assess whether the fibrin gel permeability depends on changes in thrombin generation potential and/or fibrinogen clotting property, different concentrations of thrombin with or without frozen-thawed platelets, serving as phospholipids, were used. The platelets and 0.05 IU/ml thrombin were added to plasma samples from patients with previous myocardial infarction. The fibrin gel permeability, expressed as Darcy constant (Ks), was decreased compared with that in controls, supporting findings about high risk of thromboembolism in this disease due to increases of thrombin activity and fibrinogen function. When 0.4 IU/ml thrombin was used in samples provided by 10 healthy individuals treated with acetysalicylic acid, Ks levels were increased during versus before therapy. Since almost no thrombin generation was found in the samples with the higher dose of exogenous thrombin, we considered that modifications in fibrinogen clotting property by acetysalicylic acid rendered the fibrin network more permeable. In summary, as the reproducibility remains satisfactory (coefficient of variation < 10%) despite aforementioned modifications in the equipment and reagents, any interested laboratory ought to be able to repeat the method. Assays of fibrin permeability in such a simple way may help to determine the fibrin clot stability in pathological/pharmacological studies, and probably serve as a tool to estimate thromboembolism risk in clinical materials, such as patients with cardiovascular diseases.