Abstract ISTH 2015
Jayaprakash KothaA, Juan Manuel CardenasA, Michael HerrA, Vinay BhalA, Mason DixonA, Melanie WhiteA, Sophie CombeB, Edmond GodfroidB, Lisa JenningsC
- A CirQuest Labs, Memphis, TN, USA
- B Bioxodes, Marche-en-Famenne, Belgium
- C The University of Tennessee Health Science Center, Memphis, TN, USA
Ir-CPI, the tick Ixodes ricinus salivary protein, is a serine protease inhibitor under development as a novel anticoagulant. Ir-CPI inhibits Factors (F) XIIa, XIa, and kallikrein generation, prolongs the activated partial thromboplastin time, and confers antithrombotic activity in preclinical models.
The study aim was to test Ir-CPI effects on platelet function using light transmission aggregometry (LTA), clot retraction, and Pselectin expression and to characterize Ir-CPI inhibition of the contact activated coagulation pathway by assessing FXa and FIXa activation time and activity.
Ir-CPI effects on LTA in response to 20 µM ADP and 1.6 mM arachidonic acid (AA) were studied using consented blood donors (n = 5). Platelet P-selectin expression was evaluated using flow cytometry (n = 4) following activation by contact pathway initiation. Ir-CPI effects on PRP clot retraction was measured (n = 5), and FXa and FIXa generation were determined by quantitative chromogenic assays (n = 3).
LTA response as well as clot retraction were unaffected by Ir-CPI pretreatment. However, the percent of P-selectin positive platelets was decreased following Ir-CPI treatment (0.5, 1, and 2 µM) by 5%, 9%, and 18%, respectively, vs. control. FXa activation lag times were prolonged to 704, 752, 826, 1013 s with increasing concentrations of Ir-CPI (1, 2, 4, 8 µM, respectively) vs. control (611 s). Similarly, FIXa activation lag times were 790, 834, 876, and 902 s, respectively, vs. 581 s (control). Also, the mean reaction rates (OD min -1) of FXa and FIXa with added Ir-CPI were decreased ~2.3-fold.
We demonstrated that Ir-CPI does not affect in vitro platelet aggregation response or clot retraction. However, Ir-CPI dose dependently blocked contact pathway mediated platelet P-selectin expression and the rate of FXa and FIXa activation and activity. This study confirms specific effects of Ir-CPI on the contact pathway of coagulation and provides a basis for monitoring Ir-CPI.