The key clinical feature of sickle cell disease is pain, which is usually experienced as coming from the bones or joints. Typically, the pain begins with little or no warning, quickly builds in intensity to a maximum and then gradually subsides and resolves more slowly. This is the, so-called, sickle cell crisis. The usual explanation for this pain is that the misshapen, sickle-shaped red blood cells (RBC’s) have a tendency to block up blood vessels, especially in the bone marrow, because they are unable to bend, twist and turn as easily as normal red blood cells. As a result the flow of blood is restricted and the cells and tissues are deprived of oxygen. The oxygen-deprived, or hypoxic, tissues die causing an acute inflammatory reaction with swelling and pain. Because the inflamed tissues are confined inside the bones, the swelling leads to the deep-seated, throbbing pain characteristic of a sickle cell crisis.
Although this explanation is basically correct we have learnt that the process of blood vessel blockage, or vaso-occlusion as it is called, is much more complicated than just the clumping together of sickled red cells – it is a multi-step, cascade reaction involving many other cells in the blood as well as the lining of the blood vessels. So, although the fundamental problem is the change in the shape of the red blood cell, these other processes are just as important in the onset of a crisis and pain. In a way this complexity is a “good thing”, because it opens up many more possibilities for intervening, and blocking or reversing the process.
The sickle cell mutation produces a change in the structure of sickle haemoglobin, so that the haemoglobin molecules, inside the red blood cells, have a tendency to polymerise or join up together. When they do this they form long, rigid, crystal-like structures, called tactoids, which distort the shape of the red cell changing it from a flexible round shape to an inflexible elongated structure.
This process of sickling happens repeatedly during the life of the red cell; in the tissues where the oxygen levels are low, the sickle haemoglobin tends to polymerise and in the lungs, where the oxygen level is high, the tactoids dissolve again. Over time, the repetition of this process irreversibly damages the red cell in different ways. Firstly, the lifespan of the red cell is limited, causing an anaemia, which is a universal feature in patients with sickle cell disease. Secondly, the red cell becomes dehydrated, due to a loss of salts and, as a result, is even more likely to undergo sickling in future and finally, it changes the structure of the red cell membrane so that the red cells become much more “sticky”, due to the exposure of adhesion molecules, such as alpha2-beta1 integrin and BCAM/LU. Increased “stickiness” means that they are much more likely to stick, or adhere, to other blood cells and to the endothelium, which is the lining on the inside of the blood vessels. So, not only are red cells containing sickle haemoglobin less flexible than normal red cells but they are also much more adherent, both features which promote vaso-occlusion.
But it isn’t only the red blood cells which are changed, the endothelium, or lining of the blood vessels, is also more “sticky” and receptive to red cell adhesion. Levels of TNF-alpha (tumour necrosis factor-alpha) and interleukin 1 are increased in sickle cell disease and these two chemicals are known to stimulate the endothelium to produce special adhesion molecules, including the selectins (P-selectin and E-selectin), vascular cell adhesion molecules (VCAM-1) and intercellular adhesion molecules (ICAM-1). What is more, production of these adhesion molecules is normally limited by nitric oxide, but levels of nitric oxide are low in sickle cell disease, so this also drives, or up-regulates, increased production of these molecules. So, in sickle cell disease both the red cells and the vascular endothelium are “sticky” encouraging blood vessel blockage or vaso-occlusion.
There is one other factor to consider and that is the other cells in the blood stream, because the white cells in the blood are also very important in promoting vaso-occlusion. It has been known since 1994 that sickle cell patients with the highest white cell counts tend to have the most pain and, in 2002, it was shown experimentally in mice, that red cells sticking to white cells was just as important in promoting vaso-occlusion as red cells sticking directly to the vascular endothlium. There are many different types of white cells in the blood but the most important ones for us are neutrophils and monocytes; why should white cells be involved in something, like sickle cell disease, which just affects haemoglobin and red blood cells?
Normally white cells “roll” along the inside of the blood vessels, patrolling the vascular endothelium, ready to move out of the blood into the tissues if there is any evidence of infection or tissue damage. There they help establish an area of inflammation, destroy the invading bacteria, remove damaged tissues and promote tissue repair.
“Rolling” is mediated by weak interactions between long chains of sugar molecules, such as PSGL-1, on the white cell surface and P-selectin on the vascular endothelium. Firm binding of the white cell to the vascular endothelium and movement out into the tissues is promoted by protein-protein interactions, between leucocyte integrins and endothelial VCAM-1 and ICAM-1. Many of these are the same chemicals involved in the interactions between the red cells and vascular endothelium. White cells captured by the endothelium will also strongly bind, or adhere to, sickle red cells; it appears that another selectin, called E-selectin, again located on the vascular endothelium, activates a chemical, called alphaM-beta2 integrin or Mac-1, on the surface of captured white cells, which targets and binds to, sickle red cells, possibly by complement components on the red cell surface.
To summarise then, vaso-occlusion is a complex process involving a cascade of different, but related reactions:
- Polymerisation of sickle haemoglobin and a change in the shape of the red cells to a “sickle-shape” which makes them less flexible and mechanically more prone to block up blood vessels.
- Damage to the surface membrane of the red cells with exposure of additional chemical groups on the surface making them more “sticky”.
- Activation of the vascular endothelium and circulating white cells with the production of increased levels of chemicals, promoting the adhesion of red cells to both of them.
- Adhesion of white cells to the vascular endothelium and eventual movement of the white cells out of the blood stream into the tissues and the activation of an inflammatory response.
All of these reactions can be observed, under the microscope, in the small blood vessels of mice, which have been genetically engineered so that they have sickle cell disease. In these experiments you can watch the sickle red cells sticking to the vascular endothelium or, more frequently, to the adherent white cells and as the red cells pile up the blood flow gradually slows down and stops. Scientists can use this model of sickle cell vaso-occlusion to test out various drugs to see which ones are successful in stopping the process. Currently there are three possible drug treatments under investigation. Two of the drugs have been around for a long time, and have been used to treat other disorders, the third drug, GMI-1070 is entirely new.
Rivipansel or GMI-1070 was specifically developed by GlycoMimetics, in association with Pfizer. It is a pan-selectin antagonist, in other words it blocks the action of all three selectins, E, L and P. The selectins are chemicals, present on the surface of white cells and the vascular endothelium, which are vital to the adhesion of sickle red cells. The drug was developed with the intention that it would be useful in the treatment of a range of inflammatory disorders, including sickle cell disease.
Initially, studies were carried out in mice where GMI-1970 was shown to reduce white cell adhesion to the vascular endothelium, promote white cell “rolling’ and also dramatically reduced the capture of sickle cell red cells by white cells. The drug was then studied in patients with sickle cell disease in crisis. 76 patients were recruited to the study all over the USA and Canada. The drug was administered in hospital by IV infusion twice a day during a painful crisis. The investigators found that the drug:
- Reduced the duration of a crisis by 28% from 144.6 to 103.6 hours, a reduction of 41 hours.
- Reduced the time to achieve a significant reduction in pain by 42% from 125.3 to 72 hours, a reduction of 53 hours.
- Reduced the length of admission by 84% from 156.1 to 72.2 hours, a reduction of 53 hours.
- Reduced overall use of intravenous opiate drugs by 83%.
These were encouraging findings but, despite the large number of patients enrolled onto the study, the improvements seldom reached statistical significance, because there was great variability in response between patients. The findings were presented at the American Society of Haematology (ASH) meeting in New Orleans in December 2013, but have not yet been published in a scientific journal. A larger study will be needed to prove unequivocally that the drug is helpful in an acute crisis
The two other drugs currently under investigation are intravenous immunoglobulin (IV Ig) and tinzaparin (Innohep), both have been used in medicine for a long time, but for other reasons.
Intravenous immunoglobulin is an established treatment in haematology for a disease called immune thrombocytopenic purpura (ITP) but also appears to block the activation of white blood cells, and in particular the activation of Mac-1, the chemical on the surface of the white cells which specifically binds to sickle red cells. Studies with IV Ig in patients with sickle cell disease in crisis have just started.
Tinzaparin is a type of heparin (LMWH), of which there are several different varieties, including enoxaparin (Clexane). These heparins are widely used as anti-clotting agents to prevent or treat thrombosis. However, they also inhibit P-selectin, which is important in mediating cell adhesion. In 2007 a study involving 253 sickle cell patients in crisis was reported from Jeddah, Saudi Arabia, which showed a remarkable benefit:
- The number of days with the severest pain score was reduced by 26% from 1.74 to 1.28 days, a reduction of 0.46 days.
- The duration of the crisis was reduced by 41% from 4.35 to 2.57 days, a reduction of 1.78 days.
- The length of hospital admission was reduced by 41% from 12.06 to 7.08 days, a reduction of 4.98 days.
Large numbers of patients took part in the study and all of these results reached statistical significance (p<0.05) but unfortunately, six years later, they have still not been confirmed by a repeat study from another centre. There are, however, plans at the moment to undertake preliminary studies with a drug called SelG1, which is a monoclonal antibody directed against P-selectin to see whether similarly encouraging results can be obtained.
There are therefore exciting possibilities here for treating an established painful crisis, minimizing the pain, reducing the need for strong pain relief and shortening hospital admissions. The option of investigating whether several of these agents combined together will have an additive benefit is appealing and will hopefully be taken forward in the near future.