A novel prothrombin time method to measure all non-vitamin K-dependent oral anticoagulants (NOACs).

BACKGROUND: There is a clinical need for point-of-care (POC) methods for non-vitamin K-dependent oral anticoagulants (NOACs). We modified a routine POC procedure: Zafena's Simple Simon™ PT-INR, a room-temperature, wet-chemistry prothrombin time method of the Owren-type. METHODS: To either increase or decrease NOAC interference, two assay variants were devised by replacing the standard 10 µL end-to-end capillary used to add the citrated plasma sample to 200 µL of prothrombin time (PT) reagent by either a 20 µL or a 5 µL capillary. All assay variants were calibrated to show correct PT results in plasma samples from healthy and warfarin-treated persons. RESULTS: For plasmas spiked with dabigatran, apixaban, or rivaroxaban, the 20 µL variant showed markedly higher PT results than the 5 µL. The effects were even more pronounced at room temperature than at +37 °C. In plasmas from patients treated with NOACs (n = 30 for each) there was a strong correlation between the PT results and the concentration of NOACs as determined by the central hospital laboratory. For the 20 µL variant the PT response of linear correlation coefficient averaged 0.90. The PT range was INR 1.1-2.1 for dabigatran and apixaban, and INR 1.1-5.0 for rivaroxaban. Using an INR ratio between the 20 µL and 5 µL variants (PTr20/5) made the NOAC assay more robust and independent of the patient sample INR value in the absence of NOAC. Detection limits were 80 µg/L for apixaban, 60 µg/L for dabigatran, and 20 µg/L for rivaroxaban. CONCLUSIONS: A wet-chemistry POC PT procedure was modified to measure the concentrations of three NOACs using a single reagent.

INR calibration of Owren-type prothrombin time based on the relationship between PT% and INR utilizing normal plasma samples.

Prothrombin time (PT) is clinically important and is used to monitor oral anticoagulant therapy. To obtain PT results in international normalized ratio (INR), the current standardization procedure is complex and involves reference reagents. The PT of diluted plasma samples can be determined with a combined thromboplastin (the Owren-type procedure), but not necessarily with a plain thromboplastin (the Quick-type procedure). Owren-type PT procedures can therefore, as an alternative to the INR calibration, be calibrated with diluted normal plasma to give PT results in percent of normal PT activity (PT%). The present study explored if a plasma-based calibration of an Owren-type PT procedure can be used to obtain results in INR. The approach was to establish a relationship between PT% and INR by multi-center analysis of 365 samples from healthy individuals and patients on warfarin treatment. INR values were obtained by manual Quick-type reference procedure and PT% values by various automated Owren-type procedures. A relationship INR = (1/PT% + 0.018)/0.028 was found. A calibration procedure, based on the relationship, was investigated. Calibrators were the median PT of 21 normal plasma at dilutions representing 100%, 50%, 25%, 12.5% and 6.25% of normal PT activity. These were assigned INR values of 1.00, 1.36, 2.07, 3.05 and 6.36. Calibration of various Owren-type assays was repeatedly performed by 5 expert laboratories during 3 consecutive years. The INR values of certain lyophilised or frozen control plasmas were determined. The frozen control plasmas had externally assigned INR values according to WHO guide-lines. Within the laboratory, CV was typically below 3%. No appreciable difference among the results of the different laboratories or the three assay occasions was found. Externally assigned and INR values were essentially identical to those found. These and other results indicated that the calibration procedure was reproducible, precise and accurate. Thus, an Owren-type PT assay can be calibrated with normal plasma samples to give results in INR and the investigated calibration procedure can be proposed for this purpose.

Effects of inhibition of P2Y(1) and P2Y(12) on whole blood clotting, coagulum elasticity and fibrinolysis resistance studied with free oscillation rheometry.

INTRODUCTION: In vivo, initial platelet activation is likely caused by platelet contacts with collagen in the subendothelium or from the small amounts of thrombin formed by the tissue factor/factor VIIa complex. Our aim was to study the coagulative role of ADP released by the platelets after activation with strong stimuli such as collagen and/or thrombin, and the relative importance of the platelet ADP receptors P2Y(1) and P2Y(12). MATERIALS AND METHODS: We used 10 Hz free oscillation rheometry to measure clotting time, clot elasticity and fibrinolysis resistance of non-anticoagulated whole blood. The platelets were activated with a collagen-related peptide (CRP), with the PAR1 thrombin receptor activating peptide TRAP-6 or by thrombin, the latter generated by small amounts of thromboplastin. To inhibit the platelet ADP receptors, we used the P2Y(1) antagonist MRS2179 and the P2Y(12) antagonist AR-C69931MX. RESULTS: Both antagonists significantly retarded the clotting induced by CRP. The effects were most pronounced with AR-C69931MX. For TRAP-6, the same trend was seen, but the retardation was only significant with AR-C69931MX. Clotting induced by small amounts of thromboplastin was not affected by any ADP-receptor antagonist. Addition of both antagonists did not change the results as compared to samples with AR-C69931MX alone. Nor did the antagonists, one at a time or in concert, effect fibrinolysis or the elastic properties of the clot. CONCLUSION: We conclude that ADP-receptor inhibition prolongs the clotting time for whole blood activated by CRP, but that it does not affect the properties of the subsequently formed coagulum.

Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood--different effects of collagen, TRAP and calcium ionophore A23187.

We have studied the effects of different platelet agonists on phosphatidylserine (PS) exposure and clotting times in blood without anticoagulants. Similar reductions in clotting time were obtained for collagen, TRAP-6 or calcium ionophore A23187 (50 micro mol/L), in spite of huge differences in PS expression [6.7 +/- 2.4%, 2.3 +/- 0.5% and 99.9 +/- 0.1%, respectively (mean +/- SD, n = 5)]. Furthermore, the clotting times were much longer for samples with A23187 exposing the same amounts of PS as samples with collagen or TRAP-6. Annexin V reversed the clotting time reduction, but could not prevent coagulation. Addition of phospholipid vesicles containing 20% PS neither affected the clotting times nor induced clotting in recalcified, platelet-free plasma. We conclude that platelet PS exposure is necessary, but not sufficient, for the coagulation amplification observed when platelets are stimulated via physiological receptors in a whole blood environment.

Surface plasmon resonance and free oscillation rheometry in combination: a useful approach for studies on haemostasis and interactions between whole blood and artificial surfaces.

In haemostatic and biomaterial research biological processes at surfaces and in the bulk phase of the surface-contacting medium are important. The present work demonstrates the usefulness of the combination of surface plasmon resonance (SPR), sensitive to changes in refractive index at surfaces, and free oscillation rheometry (FOR), sensitive to rheological properties of the bulk, for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma and coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with different appearance than plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood during coagulation, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyl- and hydroxyl-terminated self-assembled monolayers (SAM) on gold and gold surfaces regenerated after coagulation were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation for these surfaces was low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated detaching of the coagulum from the surface. The combination of SPR and FOR is well suited for studies of cell- and protein-surface interactions and simultaneous bulk processes. Possible applications are investigations of blood cell defects in patients and monitoring of native whole blood interactions with artificial surfaces.

Comparative studies with surface plasmon resonance and free oscillation rheometry on the inhibition of platelets with cytochalasin E and monoclonal antibodies towards GPIIb/IIIa.

In the haemostatic system a multitude of processes are intertwined in fine-tuned interactions that arrest bleeding, keep the circulatory system open, and the blood flowing. The occurrence of both surface and bulk interactions adds an additional dimension of complexity. These insights have led to the belief that global overall procedures can inform on the likely behaviour of the system in health and disease. Two sensing procedures: surface plasmon resonance (SPR), which senses surface interactions, and free oscillation rheometry (FOR), which senses interactions within the bulk, have been combined and evaluated. The contribution of blood cells, mainly platelets, to the SPR and FOR signals was explored by simultaneous SPR and FOR measurement during native whole blood coagulation, accelerated via the platelets through addition of SFLLRN peptide and inhibition of platelet aggregation with abciximab (ReoPro) and of shape change with cytochalasin E. The SPR technique was found to be sensitive to inhibition of blood cell functions such as adhesion to and spreading on surfaces, as well as platelet aggregation. SPR seemed not to be directly sensitive to fibrin polymerisation in coagulating whole blood. The FOR technique detected the coagulation as a bulk phenomenon, i.e. the gelation of the blood due to fibrin formation was detected. The combination of SPR and FOR may therefore be suitable for studies on blood cell functions during coagulation.

The role of platelets in blood coagulation--effects of platelet agonists and GPIIb/IIIa inhibitors studied by free oscillation rheometry.

We have studied the contribution of platelets to the coagulation of plasma and the effects of activation or inhibition of platelets on the coagulation process in unanticoagulated fresh whole blood (subsequently termed native blood). For this purpose, we have used a free oscillation rheometer (FOR), the ReoRox4, a new instrument that enables noninvasive viscoelastic measurements of clot formation in plasma and whole blood. Platelets appear essential for the initiation of coagulation if no activating surface is present. We prepared platelet-free plasma by quick centrifugation and filtration of native blood, which did not coagulate if stored in plastic containers at 37 degrees C but clotted if transferred to glass containers. Addition of platelet agonists, such as collagen or the thrombin receptor agonist peptide, SFLLRN, significantly accelerated the clotting of native blood and also changed the rheometer curve appearance, accelerating both onset and completion of clot formation (i.e. fibrin gel formation). Inhibition of platelet glycoprotein (GP) IIb/IIIa with the peptide-derived compound MK-852 or the antibody-derived abciximab (Reopro) prevented clot retraction and prolonged the clotting time with SFLLRN. In collagen-stimulated samples, MK-852 accelerated clotting but delayed completion of clotting while abciximab prolonged both clotting time and completion of clotting. To our knowledge, this is the first report showing that activation of platelets in native whole blood shortens the coagulation time ex vivo. It also describes a new instrument that enables studies of the viscoelastic properties of a forming whole blood clot.