Thrombodynamics, a new global coagulation test: Measurement of heparin efficiency.

The actual coagulation status may be reliably measured using only highly sensitive global functional tests; however, they are not numerous and all of them have disadvantages. Thrombodynamics (TD), a novel global coagulation test, is sensitive to hypo- and hypercoagulable states. The main properties of this test were investigated, and its capabilities for hemostasis analysis were verified through pharmacodynamic monitoring of the most widely used anticoagulants, heparins. The anticoagulant effects in the plasma of donors (n = 20) and patients after hip replacement (n = 20) spiked with unfractionated heparin or enoxaparin were measured in vitro to eliminate the influence of pharmacokinetic factors. Sensitivity for heparins was compared for activated partial thromboplastin time, thrombin generation tests and TD. TD was shown to reliably characterize the pharmacodynamics of any heparin in the entire range of its prophylactic and therapeutic concentrations. Inter-individual variability for the anticoagulant action of heparins was also calculated using the TD data. This variability did not differ between the investigated groups and did not exceed 12% and 20% for the stationary clot growth rate in the presence of unfractionated heparin and enoxaparin, respectively. That finding was in accordance with the values determined earlier using the thrombin generation test. The study results showed that TD has advantages over the other global methods of coagulation analysis. These advantages are good standardization, high reproducibility, independence of the parameter values from patient age and gender, and a narrower parameter distribution in a normal population. These results indicate that TD is a promising universal assessment method that improves the quality of hemostasis analysis because it more reliably detects deviations from the parameters' reference values.

Classic and Global Hemostasis Testing in Pregnancy and during Pregnancy Complications.

Pregnancy is associated with a significant procoagulant shift in the hemostatic system balance as well as other metabolic changes. Pregnancy can thereby provoke manifestation of otherwise dormant disorders of hemostasis (e.g., thrombophilia), or even cause new, pregnancy-specific disorders (e.g., HELLP syndrome). Application and interpretation of laboratory assays of hemostasis in pregnancy is particularly challenging, because normal physiological ranges are no longer applicable, and because the most dangerous and complex changes are not detected by classic routine coagulation/platelet assays. New global assays of coagulation and of platelet-dependent hemostasis appear to be promising in this respect, but are still far from clinical practice and rarely appear in current patient management guidelines. These global assays require a high level of research to identify their relationship to clinically significant outcomes. Here, we review the state-of-the-art knowledge of the molecular changes in the hemostatic system in normal pregnancy and during pregnancy-related complications (preeclampsia, thrombotic microangiopathies, antiphospholipid syndrome, etc.). We also discuss the sensitivity of various classic and innovative assays to these pregnancy-associated changes, and describe current and potential future applications of these assays in meeting specific clinical needs.

New Infestin-4 Mutants with Increased Selectivity against Factor XIIa.

Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 µM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.

Effect of pre-analytical conditions on the thrombodynamics assay.

INTRODUCTION: Standardization of pre-analytical conditions is the obligatory step for all potential diagnostic tests. Spatial clot growth (Thrombodynamics) is a new global hemostasis assay that considers spatial organization of coagulation. The principal parameter is rate of fibrin clot growth from the tissue-factor coated surface. In this work we studied the pre-analytical variables of Thrombodynamics assay that include conditions of blood collection, sample preparation and storage. MATERIALS AND METHODS: Blood of apparently healthy volunteers was used. Eight types of citrate blood collection tubes were tested, centrifugation conditions for plasma preparation were evaluated and impact of plasma freezing/thawing was tested. RESULTS: Among the blood collection tubes tested, BD Vacutainer glass tubes showed a significantly higher clot growth rate compared to plastic tubes. There was no difference between 3.2% and 3.8% of sodium citrate. For plasma preparation, a single 15 min centrifugation at 1,600 g shows significantly increased clot growth rate compared to plasma obtained by two sequential centrifugations (15 min 1 600 g, 5min 10,000 g). There was no significant difference between 1,600 g and 2,100 g if the second centrifugation was performed. For the second centrifugation there was no difference between 20 min at 1 600 g and 5 min at 10,000g. Frozen-thawed plasma showed increased clot growth rate compared to fresh plasma. CONCLUSION: The data represent the necessary steps for the standardization of Thrombodynamics assay and for the formulation of the operating guide.