Flip or Flop: Calcium activated chloride channel in the phosphotidylserine flip: Superactivated platelets and POAG

Abstract

Purpose : Primary open-angle glaucoma (POAG), Alzheimer’s diseases (AD), and adult macular degeneration (AMD) are common neurodegenerative diseases. Beta-amyloid (βA) deposition is widely believed to be a key pathological event of these diseases. Our laboratory has identified microvascular abnormalities and superactivated platelets (SAPs) in these diseases. The phosphatidylserine (PS) flip in which the inside of the plasma membrane everts to the outside is required for SAPs formation and occurs in blood coagulation, rod outer segment shedding, and removal of apoptotic cells.

Methods : Using a proteomic approach, a calcium activated chloride channel, TMEM16F, was identified as candidate “flippase.” Whole blood was obtained from controls after informed consent approved by the Univ. Il. at Chicago IRB, anticoagulated, centrifuged at 200xg for 15 min to isolate plasma rich platelets (PRP), washed with Tyrode’s buffer and resuspended in FACS buffer. PRP (1 × 10-8/mL) was challenged with the calcium activated chloride channel inhibitor A01 (Sigma-Aldrich; 6-tert-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid) from 0.2 to 20 μM or vehicle control and activated with dual platelet agonists thrombin and convulxin at 37°C. PS flip was labeled with FITC Annexin A5 and analyzed with a Cyan ADP flow cytometer and Flow C6 software set to exclude microparticles. Fluorophore conjugated antibodies were used to identify platelet status: CD41-PE (Invitrogen) for all platelets; PAC-1-FITC (BD Biosciences) for activated platelets; streptavidin-APC (BD Biosciences) for SAPs. In addition, a cone plate rheometer measured viscosity changes induced by thrombin in the presence of the inhibitor A01.

Results : The application of A01 prevented SAPs in a dose dependent manner (Figure 1; n=3, p-value = 0.004). A01 also prevented thrombin induced low shear rate viscosity and fibrin aggregation which is a proof of concept that the “flip” is required for blood coagulation.

Conclusions : TMEM16F activity is required for platelet PS flip. The flippases are transmembrane lipid transporter proteins and aid in the movement of phospholipid molecules, also known as a "flip-flop" transition. Notably, the PS flip may serve as an early indicator of apoptosis and has been observed in neurons as a response to βA, thought to be a primary factor in POAG, AD, and AMD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Figure1. A01 inhibition of SAPs