Sticky red cells – learning about the glue

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.

This is the simplistic view of how sickle cells block the flow of blood. The stiff and inflexible sickled red cells cannot pass through small blood vessels and  mechanically pile up in a tangled mass blocking the blood vessel.

This is the simplistic view of how sickle cells block the flow of blood. The stiff and inflexible sickled red cells cannot pass through small blood vessels and mechanically pile up in a tangled mass blocking blood flow.

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 is an electron micrograph of a sickle  red cell which has broken open to reveal the rigid rods or tactoids of sickle haemoglobin

This is an electron micrograph of a sickle red cell which has been broken open to reveal the rigid rods or tactoids of sickle haemoglobin

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.

Red cells are not the only cells in the blood; different types of white blood cells help to protect us against infection and platelets help the blood to clot. The vascular endothelium is the lining on the inside of the blood vessel.

Red cells are not the only cells in the blood; different types of white blood cells help to protect us against infection and platelets help the blood to clot. The vascular endothelium is the lining on the inside of the blood vessel.

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.

This diagram shows white cells rolling along the vascular endothelium using weak interactions with selections before adhering firmly via integrin receptors, before leaving the blood vessel to migrate out into the tissues.

This diagram shows white cells rolling along the vascular endothelium using weak interactions with selectins before adhering firmly via integrin receptors and leaving the blood vessel to migrate out into the tissues.

“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:

  1. 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.
  2. Damage to the surface membrane of the red cells with exposure of additional chemical groups on the surface making them more “sticky”.
  3. 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.
  4. 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:

  1. Reduced the duration of a crisis by 28% from 144.6 to 103.6 hours, a reduction of 41 hours.
  2. Reduced the time to achieve a significant reduction in pain by 42% from 125.3 to 72 hours, a reduction of 53 hours.
  3. Reduced the length of admission by 84% from 156.1 to 72.2 hours, a reduction of 53 hours.
  4. 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:

  1. 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.
  2. The duration of the crisis was reduced by 41% from 4.35 to 2.57 days, a reduction of 1.78 days.
  3. 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.

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About rogerjamos

I am a consultant haematologist who has worked in Hackney, London, UK with patients who have sickle cell disease for many years. Knowledge is power; the hope is that this blog will empower patients by putting them in touch with contemporary research into sickle cell disease and facilitating informed discussion on the issues raised. Dr Roger Amos MA, MD, FRCPath
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10 Responses to Sticky red cells – learning about the glue

  1. Lisa Rose says:

    Hello again Dr. Amos,
    Another excellent post- I learn so much each time! As usual, I have some questions for all of us non-medical readers. In addition, this time, I also have some comments.
    Questions:
    1) You mentioned that the sickling frequently starts in the bone marrow- is this almost immediately after the RBC is produced OR does the RBC ever travel back to the bone marrow after it’s initial release?
    2) If the RBC typically sickles when it is losing oxygen down towards the tissues and away from the lungs, is it safe to assume that most sickling occurs in areas of the body that are just before the RBC makes it back to the lungs (ie- in areas that have the lowest oxygen because the RBC is just about due for a “refill”?
    3) Why do the tactoids dissolve in the lungs? Is it the oxygen that causes this or some other chemical reaction? And HOW does it dissolve (perhaps another blog post!)
    4) Why does the repetition of sickling and dissolving cause a loss in salt?
    5) The white blood cell talk was a bit technical- is it accurate to say that as the white cells began to leave the endothelium to repair tissues, but they were unable to exit due to the increase in stickiness of the endothelium, thus trapping them and increasing the area for which RBCs could in turn stick to?

    Comments on New Stickiness Treatments:
    I have heard of all these, but was recently made aware of a 4th option during our national research conference down in Miami last month. Have you heard of MST-188? It is in phase 3 clinical trials and based on what I have read (which again is very technical and much more targeted to doctors) it adheres to damaged cells, thus increasing the squishiness again and ultimately decreasing pain through a single dose. For non-medical readers they refer to it as the WD-40 for your blood, ie- it loosens things up so they can flow better. Studies so far look promising,

    I loved your comment on multimodal treatments- imagine the possibilities if even 2 of these drugs could be used in tandem to cut pain crises in half or more!

    • rogerjamos says:

      I will do my best to answer the questions.
      1. Red blood cells are produced in the bone marrow and released into the blood as it flows through the bone marrow sinusoids. Sickling, the process whereby the sickle haemoglobin polymerises and distorts the normal shape of the red cell into a rigid sickle shape, is supposed to happen in those parts of the circulation where the levels of oxygen are lowest. This is traditionally thought to be in the small venules, or veins, which drain the capillary beds, because most of the oxygen will have been extracted from the blood by the tissues as the blood passes through the capillary network. This sickling then leads, through a complex process as described in the blog, to blockage of the blood vessel, or vaso-occlusion. The red blood cells never return to the bone marrow TISSUE but do circulate through the bone marrow, supplying oxygen and other nutrients in the same way as they do through all other tissues and organs of the body. In most cases sickle cell pain is perceived as coming from the bones and joints, so we can assume that most episodes of vaso-occlusion will affect the circulation through the bone marrow. Although all tissues of the body can be affected by vaso-occlusion the bone marrow seems to be the most commonly affected; as far as I understand this fact is unexplained.
      2. Yes, quite right, as explained above.
      3. It is only deoxy sickle haemoglobin which is capable of forming tactoids. Once the red cells enter the circulation of the lungs they are exposed to high levels of oxygen, from the air breathed in, and the sickle haemoglobin combines with the oxygen to from oxy sickle haemoglobin. At this point the tactoids dissolve, a bit like a crystal of salt dissolving in water and the red cell regains it’s normal shape again.
      4. Red cells can undergo a limited number of episodes of sickling before they are so damaged by the repeated process that they are unable to return to a normal shape again.At this point the red cells are thought to become irreversibly sickled cells and dense cells. The damage to the red cells by repeated episodes of sickling affects mainly the membrane around the cell. All cells have a tendency to lose potassium and gain sodium, but the action of a pump mechanism (the sodium – potassium pump) in the membranes of all living cells ensures that most sodium is kept outside cells and a high concentration of potassium is maintained inside cells. Damage to the red cell membrane means that potassium, chloride and water are lost from the red cells, which also gain sodium to a lesser degree. The dehydration and consequent increased concentration of sickle haemoglobin increases the tendency of sickle haemoglobin to sickle. You may hear about two other pump mechanisms in the red cell membrane, the Gardos channel, which facilitates the movement of potassium out of the cell in exchange for calcium moving inwards, and the potassium – chloride co-transporter (KCl co-transporter) which moves potassium, in association with chloride, out of the red cell. Both of these channels seem to be activated by membrane damage and therefore promote the loss of potassium and water. It was once hoped that drugs which inhibited these latter two pumps might be beneficial in sickle cell disease but these hopes have not been realised to date.
      5. Not quite! The white cells are still able to leave the vascular space and migrate out into the tissues to do their thing. The point is that activation of the white cells provides a sticky surface onto which sickle red cells can adhere, just like they adhere directly to the lining of the blood vessels, or endothelium.
      I will try and put together a blog about MST-188 – I like the idea of WD-40 for the blood!

      • Lisa Rose says:

        Greetings Dr. Amos! I have been so educated and inspired by your posts that I wanted to take the information down a notch for those that know NOTHING about the medical components of Sickle Cell. You know the old saying, “The best way to internalize information is to teach it to others.” Well that is exactly how I view the learning that I receive here. You have taken information to the next level and allowed a non-medical person such as myself the opportunity to understand and converse using technical, medical terminology. Thank you for that.
        My task- take your information and create a format that all adult learners can access, even if they don’t read.
        HOPE is doing a 30 day educational video challenge for World Sickle Cell Day, but this last post Healthy vs. Sickle, Part 1 is my attempt to teach the world about the new information I’ve learned from you- sickle cells aren’t born deformed, how tactoids are formed and what they are exactly, and how the stickiness occurs. If you have time, check it out here. http://youtu.be/qzHvrJsCdIk I would love to make sure I’m on the right track.

      • rogerjamos says:

        Hi Hope for SCD – thank you for your comments. I have had a look at the video and I am truly amazed by the sophisticated props that you have! Seriously though the video is great and does an excellent job of conveying very complex ideas in a simple and accessible way. I will look forward to the follow ups. Just one small thing; sickle haemoglobin is actually quite good at binding and transporting oxygen, at least as good as adult haemoglobin. True, when the tactoids form the sickle haemoglobin is in the deoxygenated state, usually after it has released it’s oxygen in the tissues. As you say in the video, as soon as the red cells reach a well oxygenated environment, namely the lungs, the sickle haemoglobin tactoids dissolve and the sickle haemoglobin goes back into solution. So rather than sickle haemoglobin tactoids not being able to carry oxygen the point is that they only form in conditions where the available oxygen is in short supply. I hope that makes sense?

      • Lisa Rose says:

        Yay, at least we were 90% on track! I’ll take it since I chose the field of education rather than medicine. OK, so is it fair to say that the sickle hemoglobin are so unstable that they have to be “holding on” to something at all times? What I mean is- after they release the oxygen, are they desperate to bond to something else, leading ultimately to the forming of tactoids? If so, are they MORE attracted to oxygen, leading to the dissolution of the tactoids?
        Also, are th sickle hemoglobin actually successful in transporting the oxygen to the needed tissue, or do they release it too soon?

  2. Ajoke says:

    It would be really nice to repeat the study with tinzaparin, sounds very promising! All the drugs mentioned are for the purpose of relieving pain , are there any new drugs available to manage dangerous complication of sickle cell?

    • rogerjamos says:

      Thank you Ajoke – I agree absolutely, one study with positive results really doesn’t mean very much. It is very important that the work is repeated by other centres and then, if the same positive results are reproduced, we would feel much more confident that what was observed the first time round was real and not just a statistical fluke. Although pain is the main symptom of vaso-occlusion, it is the same process of blood vessel blockage, leading to impaired blood flow and oxygen deprivation which is thought to underlie the damage to many other organs in sickle cell disease giving rise, in time, to the long term complications of the illness. Therefore if we had useful drugs to prevent or inhibit vaso-occlusion they would be useful in controlling both pain and the long term complications.

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