Correlation Between the VerifyNow P2Y12 Assay and the Newly Developed APAL System in Neuroendovascular Patients.

OBJECTIVE: Light transmission aggregometry (LTA) is the gold standard method for assessing platelet function. Recently, a new parameter called adenosine diphosphate (ADP)-induced platelet aggregation level (APAL) was developed to aid interpretation of LTA results. APAL is a score calculated based on platelet aggregation patterns upon exposure to 1 µM and 10 µM ADP and is determined using an automated coagulation analyzer. We compared APAL and VerifyNow P2Y12 assay for neuroendovascular patients. METHODS: 42 patients who have received antiplatelet therapy were studied. Platelet function tests were performed on CS-2400 for APAL and VerifyNow P2Y12 assay was used for P2Y12 reaction unit (PRU) and % inhibition. RESULTS: Moderate correlations were observed between APAL and PRU (r=0.64, p<0.001) and between APAL and % inhibition (r=-0.74, p<0.001). The optimal threshold for APAL was 8.2 for PRU (threshold=240) and 8.1 for % inhibition (threshold=26%). The percentage of agreement between the above thresholds was 90.9% between PRU and APAL and 77.3% between % inhibition and APAL. CONCLUSIONS: The APAL system exhibits moderate correlation with PRU and % inhibition. APAL testing is a good choice for a clinical laboratory already in possession of Sysmex CS series analyzers. In this setting, APAL testing can significantly decrease the cost of platelet function testing for patients on antiplatelet therapy.

Evaluation of the Newly Developed Adenosine Diphosphate-Induced Platelet Aggregation Level System in Aggregometer on Automated Coagulation Analyzer.

BACKGROUND: Light transmission aggregometry (LTA) is the gold standard for platelet function assessment. The automated coagulation analyzer from Sysmex that performs LTA offers the advantage of being a walk-away technology. Recently, a new parameter "ADP-induced platelet aggregation level (APAL)" was developed to support the interpretation of results. APAL is calculated as a score from 0.0 to 10.0 based on platelet aggregation patterns with 1 and 10 µM adenosine diphosphate (ADP). Here, the basic performance of the newly developed APAL system and comparison with the maximum aggregation rate of ADP (ADP-MA) was evaluated. METHODS: The within-run precision was calculated by conducting five replicate analyses of the platelet-rich plasma (PRP) from healthy volunteers and 0.05 µM of cangrelor-spiked PRP. Cangrelor is a P2Y12 inhibitor that does not require liver CYP activation. The reference interval was calculated from the results of 67 healthy volunteers. The effect of the antiplatelet P2Y12 agent was evaluated using several concentrations of cangrelor. A comparative study was performed using 103 PRP samples with different levels of aggregation. Each test was analyzed with both APAL and ADP-MA. RESULTS: The percentage coefficient of variation in within-run precision was within 7% for APAL and 10 µM ADP-MA. Reference interval of APAL and 10 µM ADP-MA was 7.1 - 10.0 and 80.0 - 99.2%, respectively. APAL signifi-cantly decreased with the addition of 0.02 µM cangrelor, while 10 µM ADP-MA was barely affected. A significant correlation was observed between APAL and 10 µM ADP-MA (r = 0.94; p < 0.0001). CONCLUSIONS: The newly developed APAL system exhibited an acceptable performance. APAL score showed a good correlation with ADP-MA and was adequate to detect the weak effect of P2Y12 inhibitors. APAL is a new platelet aggregation scoring system with the potential to monitor the effects of P2Y12 inhibitor over a wide range.

[Accuracy and reliability of international normalized ratio (INR) in monitoring of warfarin--comparison in the sensitivity to extrinsic coagulation factors between PT (INR) and TT (INR)].

Prothrombin time (PT) is widely used as the monitor of oral anticoagulant therapy using ISI/NR system which WHO recommended. However, a main clinical usefulness of an Owren-type combined reagent (TT) also remains in the monitoring of warfarin in Europe and Japan. Recently, a TT reagent utilizing a recombinant bovine tissue factor (TF) expressed by silkworm-baculovirus system has been developed. The purpose of this study is to investigate the sensitivity to coagulation factors (FII, FV, FVII, FX) of PT (rabbit brain and recombinant human) and TT (bovine brain and recombinant bovine) in INR, and the correlation among those reagents using 53 plasma samples from patients treated with warfarin. The INR results were calculated using a certified INR calibrator, "AK-CALIBRANT", according to the method of "Direct" INR determination. The sensitivity to FII, FVII and FX of those reagents results similar behavior in INR, but the sensitivity to FV of PT reagents were generally higher than that of TT reagents. The correlation coefficient between recombinant PT and recombinant TT was 0.979. There were a good agreement between two TT reagents (bovine brain and recombinant bovine) in INR (r = 0.998). It was apparent that a variance between PT and TT was dependent on the sensitivity to FV level during the course of warfarin treatment in a clinical case. In conclusion, PT and TT reagents gave generally acceptable correlation in INR, and our results indicate that the Owren-type TT reagent is also well suited for monitoring warfarin using local INR calibration according to WHO recommendation.

A Synechocystis homolog of SipA protein, Ssl3451, enhances the activity of the histidine kinase Hik33.

In the cyanobacterium Synechocystis sp. PCC 6803, the histidine kinase Hik33 regulates the expression of several stress-inducible genes. Recently, a yeast two-hybrid screen revealed a specific interaction between Hik33 and a small protein, Ssl3451. To investigate the function of Ssl3451, we developed an assay to monitor the autophosphorylation of Hik33 in vitro. Addition of Ssl3451 to the reaction mixture dramatically enhanced the autophosphorylation activity of Hik33. Pulse-chase experiments revealed that Ssl3451 stimulated the autophosphorylation of Hik33 but did not affect its dephosphorylation. These findings indicated that Ssl3451 might be an activator of Hik33. When the amount of Hik33 was kept constant and the amount of Ssl3451 was increased in the reaction mixture, the extent of autophosphorylation of Hik33 reached a plateau when equimolar concentrations were present, suggesting that Ssl3451 enhances the activity of Hik33 by associating with it with a 1 : 1 stoichiometry. Disruption of the gene for Ssl3451 resulted in increased expression of the hliB gene, which is induced by Hik33 under standard growth conditions, but it did not affect the levels of the hliB mRNA at low temperature. Together, these results suggest that Ssl3451 might enhance the activity of Hik33 both in vitro and in vivo.