Platelet Problems in Cystic Fibrosis

Long Read, written by Malgorzata Urbacz

Researchers based at UCSF, San Francisco, has identified platelets as a cause of lung inflammation in cystic fibrosis, shaking up long-held ideas about the disease.

Cystic fibrosis (CF) is a genetic disorder that affects mainly the lungs, though it also causes abnormalities throughout the digestive and reproductive systems. The gene responsible for this disease is Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), whose role is to transport chloride ions across epithelial cell membranes. Usually, CFTR transports chloride into the mucus, which then attracts the movement of water into the mucus so that it is at a consistency that is easily expelled from the lungs. When CFTR is mutated, chloride ions are unable to be transported into the mucus, therefore water does not follow, and the mucus becomes thick and sticky. The most common mutation in CFTR is a missing phenylalanine at position 508 (F508), which disturbs the folding of the channel and renders it non-functional.

The build-up of mucus in the lungs encourages frequent respiratory infections, as bacteria become trapped and readily multiply within the airways. Mucus can also build up in pancreatic ducts and block the release of digestive enzymes into the lower intestine, leading to malnutrition and diabetes. All these effects combined lead to a severely reduced life expectancy, with only half of sufferers reaching the age of 40.

Inflammation Issues

The main cause of death in CF patients is the rapid decline in lung function due to damage caused by excessive inflammation of the airways. This inflammation is characterised by a large influx of neutrophils, a type of white blood cell which migrates to the airways in response to a developing infection. Upon their arrival at the lungs they release damaging chemicals such as oxygen species and proteolytic enzymes, which damage the lung tissue until it is not able to support life.

For a while now, researchers have fought hard to identify the cause of this inflammation, in the hope that they can develop ways to prevent it from happening. Up until recently it was thought that neutrophils were the culprit, releasing an abnormal amount of cytokines which overloaded the already suppressed epithelial host defences, and damaged the cells. Recent research from The University of California, San Francisco, has identified that platelets stand behind this deadly lung inflammation.

Platelets are key in inflammation responses in a huge variety of diseases, releasing chemical mediators to recruit white blood cells to the site of infection or injury. Some of these chemicals, known as chemokines and cytokines, also increase vascular permeability, promoting the flow of blood and lymph to the site of inflammation. In order for platelets to release these chemical mediators they must first be activated, by an influx of calcium into the cell, through the calcium channel Transient Receptor Potential Cation Channel Subfamily C Member 6 (Trpc6).

The researchers worked with knockout mice, which completely lack the CFTR gene. These mice showed an increase in lung inflammation after they were infected with Pseudomonas aeruginosa bacterium, a common pathogen that infects CF patients. Later, they introduced a cell-specific CFTR gene deletion into the mice, either in platelets or neutrophils.

The Platelet Problem

The mice which lacked CFTR in platelets had a significantly higher bacterial load and increased lung inflammation than those with a CFTR missing from neutrophils. Additionally, they found aggregate bundles of neutrophils that had become stuck to platelets in the mice with a CFTR deletion in the platelets, but not in the neutrophil-specific knockout. The researchers then compared platelets from healthy volunteers and CF patients and found that the platelets from the patients were in a higher state of activation, meaning they were more likely to form aggregates with neutrophils. The researchers were able to identify that the calcium channel Trpc6 was responsible for the increased inflammation. Previous studies have shown that airway epithelial cells taken from CF patients have an abnormally large intracellular calcium concentration, which was initially thought to be due to a gain-of-function mutation of Trpc6. Co-immunoprecipitation experiments pull out groups of proteins to see which of them directly interact and these showed that CFTR was attached to Trpc6, and that it was actually the F508 mutation in CFTR causing increased calcium influx. Researchers were able to demonstrate this by reversing the CFTR F508 mutation to show the intracellular calcium concentration returning back to normal. 

graphical abstract

The scientists from California found that the deletion of CFTR in platelets caused an increase in calcium influx into the platelets themselves through Trpc6, just as was seen previously in airway epithelial cells. Platelet activation, aggregation, and thrombus formation all require the conduction of calcium ions through Trpc6. This explains why, with an abnormally large intracellular calcium concentration, the platelets were in a higher state of activation and formed aggregates with neutrophils. As they were stuck, the neutrophils were unable to reach the infection and clear the bacteria, hence why the mice with a CFTR deletion in the platelets had a larger bacterial load. The findings of this study suggest that our knowledge of CF disease development and progression may, in some parts, be incorrect. It was generally accepted that lung inflammation in CF occurred in response to an infection, however, these results provide evidence that lung inflammation may be a primary abnormality of the disease. It may be that the mutated CFTR is directly influencing Trpc6 and causing lung inflammation, even in the absence of bacterial infection.