PB0881 - Calcium-and integrin-binding protein 2 negatively regulates in vivo thrombosis and platelet functions by attenuating S1P production

Background: CIB family consists of four members, CIB1, CIB2, CIB3, and CIB4. CIB1 positively regulates platelet function. The expression and function of other members in platelets is not known.

Aims: To evaluate the function of CIB2 in platelets.

Methods: We generated Cib2flox/Pf4cre+ mice. Cib2flox/Pf4cre- mice were used as controls. In-vivo tail bleeding, microfluidic assays, and tMCAO model of stroke as well as aggregation, Ca2+ levels, western blotting, and RT-PCR were performed.

Results: We found that CIB2 is expressed in platelets. MK/platelet specific loss of Cib2 enhances hemostatic and thrombotic function of platelets which is opposite to Cib1 ablation thus ruling out the proposed compensatory role for Cib2. The absence of Cib2 results in increase in platelet aggregation (P < 0.05) and intracellular calcium rise when stimulated with low concentrations of agonists which was overcome by increased agonist concentration. Additionally, the in-vitro thrombus formation was significantly (P < 0.05) enhanced in Cib2-null platelets. The observed gain of function is due to enhanced platelet signaling and not due to increased expression of surface receptors. Furthermore, deletion of Cib2 results in enhanced cerebral infract size, which is opposite to that observed in Cib1 KO mice. CIB1 interacts and translocate sphingosine kinase 1 (SK1), a key enzyme involved in S1P synthesis. CIB2 on the other hand inhibits SK1 translocation since it lacks the calcium-myristoyl switch. It is therefore possible that in the absence of Cib2 increased S1P is generated causing enhanced platelet function. Inhibition of S1PR2, a major S1P receptor using JTE-013, we found that the agonist-induced hyper-aggregation and increase in calcium level in Cib2flox/Pf4cre+ platelets were reduced to control levels. Furthermore, treatment of Cib2flox/Pf4cre+ mice with JT3-013 reduced the infract size to control level.

Conclusion(s): These in-vivo and in-vitro results suggest that Cib2, an endogenous suppressor of SK1, negatively regulates thrombosis and stroke by attenuating S1P production.