“The two magic words of hemostasis: amplification and regulation"

November 9, 2016

Professor Joost Meijers is manager of the Plasma Proteins department of the Research Division of Sanquin. He started his career at Utrecht University, became professor at the AMC Amsterdam and joined Sanquin in 2014. Besides doing research he committed himself to teaching and was involved in numerous committees in the field of hemostasis and fibrinolysis. He is currently chairman of the ISTH SSC Subcommittee on Factor XI and the Contact System, Secretary/Treasurer of the International Society on Fibrinolysis and Proteolysis, and is member of the Executive Board of the Dutch Thrombosis Foundation.

You moved to Sanquin in 2014. How do you like it here?

The number of scientists working on hemostasis in the Netherlands is rather small. At Sanquin I am part of an entire team of scientists all dedicated to this field of research. It feels like coming home. Besides, there is a great deal of biochemical expertise throughout the institute. My research group runs like a well-oiled machine. People all work from an inner motivation. That means that although I’m the manager I can still spend most of my time on research.

Why are so many factors and proteins involved in the homeostasis process?
This can be explained by two magic words: amplification and regulation. When we bleed the body should respond with a powerful reaction to stop the bleeding. Therefore we need amplification. This is necessary for swift coagulation at the site of the injury. But this process could derail tremendously. All kinds of regulatory proteins take care of keeping the coagulation process in control. Nevertheless, in the Netherland 400,000 people need anticoagulants to prevent thrombosis. These medications are also prescribed to hospitalized patients. Doctors of nearly all medical disciplines need to deal with coagulation abnormalities. Thrombosis can be seen as too much coagulation at the wrong place at the wrong time.

What is the importance of the contact pathway of coagulation?
The proteins of the contact system, factor XI, factor XII, prekallikrein and high-molecular weight kininogen, were discovered in the 1950s and 1960s. Blood drawn from patients with a factor XI deficiency shows a prolonged clotting time. Obviously, at the time it was thought that the contact system was necessary for proper coagulation. But curiously, those patients hardly have any bleeding problems. The coagulation delay is foremost an in vitro phenomenon. Now we think that the contact system contributes to thrombosis. For instance knock-out mice deficient in one of the contact proteins are protected against thrombosis.

What will be the anticoagulant of the future?
Current medicines like heparin, vitamin K antagonists or direct oral anticoagulants all have one important side effect: bleeding. With my team I focus on development of inhibitors of factor XI. That could be a safe alternative for the currently available drugs. You could think of monoclonal antibodies or small molecules. To develop inhibitors in our lab we want to know everything about factor XI. How does it work? How does it react with factor XII and thrombin? When we understand which part of the molecule is important for its function we can better attack the protein. But we would also like to develop an improved factor XI protein, one that stimulates coagulation. That could help patients with bleeding disorders like hemophilia.

What would you like to achieve at Sanquin?
Recently the Molecular Cell Biology department, with their expertise on blood vessels, was integrated in our department. I want to combine both research fields. It is the blood vessel wall where bleeding, coagulation and thrombosis all start. Endothelial cells play an important role in hemostasis. We can learn a lot from each other and together we can do high quality research. I hope I can obtain a European grant for continuing fundamental research on factor XI, in the end hopefully leading to development of pro- and anticoagulant medication. We already work together with a Dutch company to identify small molecules that stimulate coagulation and may be useful in patients with bleeding disorders.

Finally, what goes through your mind when you bleed?
When I accidentally cut my finger I can only see the beauty in the blood. I think of the interplay between systems that cause my blood to clot. That is, of course, before putting a sticking plaster on my finger.

Published at Interview Blog Sanquin Talks

Theo Rispens: “I try to envisage how molecules interact with each other”

February 12, 2015

Theo Rispens

Theo Rispens is group leader at the department of Immunopathology Research. His research topics cover all aspects of antibodies, from structure-function analysis of IVIg to regulation of antibody production. This includes immunogenicity of biologicals.

You have studied ‘hard-core’ chemistry, what brought you here at Sanquin?
“My PHD project involved research in the field of physical chemistry. Although the science was interesting, I missed a connection with society. At Sanquin I got the opportunity to combine basic chemistry with translational, patient-directed research. I started with a project that was fairly physical-chemical in nature, studying the stability of therapeutic immunoglobulin preparations and developing methods to do so. Within Sanquins bio-medical work field, my background in chemistry helps me to perceive biological processes from a molecular point of view. I try to imagine how fast individual molecules move about and how they interact with each other. An immune response is all about recognition of dangerous pathogens, which ultimately involves interaction and binding of molecules, like antibodies to antigens.

What is so fascinating about antibodies?
“Antibodies are molecules that show endless structural variations. But what they all have in common is their bridging capacity. Antibodies span bridges between pathogens and a variety of immune cells and thereby one way or another activate our immune system. What intrigues me most is how B-cells randomly combine a set of gene fragments to produce this enormous diversity of antibodies that can fulfill a variety of functions. The immune system is able to generate antibodies against virtually any thinkable antigen. Depending on their classes or subclasses or glycosylation, antibodies are even capable to dampen the immune reaction, like for instance IgG4 does. Sometimes we produce harmful antibodies, as we see in autoimmune diseases. In the clinic antibody-based therapy has become established these days. In various medical fields a wide variety of therapeutic antibodies is being used, all with their own unique features.

What makes IgG4 so special?
Antibodies have two identical Fab arms that bind antigen. However, “IgG4 exchanges half-molecules in the body, and that way obtains two different Fab arms. Therefore, unlike IgG1, IgG4 binds to its target with only one Fab-arm, cannot crosslink and therefore cannot form large immune complexes. That’s one reason why IgG4 is a poor trigger of any effector function. It is less effective in activating the rest of the immune system. You could see IgG4 as a mild antibody. IgG4 can block the antigen, but not trigger further action. When we are exposed to an antigen for a long time, for instance to a food antigen, the immune system often switches to IgG4 production, which may dampen the immune response and prevent overacting by the immune system. This is another form of regulation of the immune response.
However, sometimes IgG4 is associated with disease, like in IgG4 related disease. This disease has only been known for 10-15 years. Many organs can be affected, including the pancreas; this type of pancreatitis is often mistaken for a tumor. The pancreas is full of IgG4-producing plasma cells, and serum IgG4 titers are often extremely high. Treatment usually consists of corticosteroids. I’d like to know whether IgG4 is the cause or the result of this disease. What is the mechanism behind this disease? Why is the body making so much IgG4, is there a particular antigen?”

How does glycosylation affect the functioning of antibodies?
“All antibodies have a glycosylation site in the Fc-part of the molecule, the tail that is involved in effector functions such as complement activation and binding to Fc-receptors on immune cells. Together with other departments of Sanquin we try to find out how various glycans affect effector functions. How and when does glycosylation of antibodies take place in our bodies? Can we design antibodies with more potent effector functions? This could be of interest for development of therapeutic antibodies. Besides Fc-glycosylation, some antibodies have glycosylation sites on the Fab–arm, depending on the amino acid composition. We know very little about this Fab glycosylation. It is feasible that Fab glycosylation could affect the binding to the antigen. We’d like to find out whether this glycosylation varies in the course of the immune response and whether it contributes to regulating this response, or may contribute to affinity maturation.”

Why do some patients make antibodies to biologicals and others don’t?
“I would like to turn the question around. Why do not all patients make antibodies to their therapeutics? A growing number of patients is treated with therapeutic antibodies and derivative molecules, for a variety of diseases. Depending on the test we use we are able to detect antibodies to some of these drugs in a majority of patients. Regardless how “human” a therapeutic antibody may be, it will always include a foreign epitope, the antigen-binding site, the idiotype, which could elicit an immune response. We think genetic components may determine whether patients produce only sub-clinical amounts of antibody, or develop a full-blown immune response, completely inhibiting their biological. It is impossible to predict immunogenicity at this moment. What we can do is optimize dosage and dosing schedule to overcome immunogenicity. Co-medication of immune suppressiva is also important in reducing the immune response.
An anti-idiotype response itself is not unheard of. All the time we generate new antibodies that our immune system hasn’t seen before. Niels Jerne won the Nobel prize in part for his antibody-network theory. He claimed that our body makes antibodies to our own antibodies. And subsequently antibodies to those antibodies. And so on. With as final result antibody responses that are regulated by (auto-)antibody responses. Because therapeutic antibodies are directed to a human target protein, the antibody response to a biological can also be interpreted as an attempt of the immune system to neutralize these autoantibodies.

How would you develop a biological that is not immunogenic?
“It is not necessary to completely eliminate immunogenicity, as long as it remains low and doesn’t affect therapy. I think a good approach would be to remove dominant T-cell epitopes from therapeutic antibodies. Though prediction of those epitopes is not flawless, we would already make a lot of progress if the worst cases would be eliminated. T-cell reactivity is something that you can test in a lab, by exposing peptides to panels of cells of various people. Furthermore, choosing antibody clones with high stability will minimize aggregation of the biological, which is a known cause of immunogenicity. If the function of a therapeutic antibody is to block a target, IgG4 would be a good choice. In theory, such an antibody might be less immunogenic than its IgG1 counterpart, because it has less options to interact with the immune system. However, this has not yet been proven.

This interview has been published before at http://sanquintalks.org/theo-rispens/

“A personalized approach to donation prevents deferrals and keeps the donor healthy.”

October 15, 2014

Katja van den Hurk

Katja van den Hurk works as a senior epidemiologist at the department of Donor Studies at Sanquin Blood Supply. Her main research topics are Donor characteristics and Donor health. Sanquin, by law the only blood bank in the Netherlands, has an extremely loyal donor population of about 2.5% of the Dutch population, that make approximately 800.000 donations per year. In the Netherlands the blood supply is dependent on voluntary non-remunerated donors.

Sanquin has organized the first European Conference on Donor Health and Management, which took place on September 3-5, 2014. What were the highlights?
“Meeting 250 enthusiastic professionals in a relatively small work field is thrilling. Current regulations around blood donation are mainly based on expert opinions instead of evidence based science. The conference program balanced between science and daily blood bank practice. Managers, recruitment professionals and scientists came together and exchanged information. One example of an interesting presentation was by Olga Flinter and Stephen Cousins. They showed how the Irish blood bank fine tunes the blood donor management by using long term and short term prediction models. We presented our own large cohort studies and saw interesting cohort data from Danish and British research groups. You could really see the field is upcoming.”

How healthy is the Dutch donor?
“The donor health check, which precedes donations, already selects for a healthy population. That makes the Dutch donor population healthier than the average Dutch population. Besides, donating blood (in the Netherlands) is pro-social behaviour, since donors are not paid for donations. This might lead to selection of a more health-conscious subgroup of the average population. ”

How do you study the effect of donation on donor health?
“The so called healthy donor effect complicates comparing donor health with the health of the general population. So we try to compare different groups within the donor population. For instance new donors versus donors with a long donation career. Or donors who donate frequently to those who donate less frequent. There are several factors that influence donation frequency independent of health-related factors, such as residence. Donors living in remote parts of the country donate blood at the mobile blood center, which travels the country with a relatively low frequency. However, the health of a rural population may be different than that of an urban population. We try to correct for factors like this as much as possible.”

Approximately 5% of the donors is deferred at the collection center because of low Hemoglobin (Hb). Does that imply they are anemic?
“No, the cut-off that is handled for Hb is well above the lower limit for normal Hb concentration, in order to protect the donor. However, we cannot exclude that donors are iron-deficient with a normal Hb. Hb is only an indirect marker for body iron, so it is possible that Hb levels are maintained before donating, while iron stores are depleted and therefore insufficient to restore Hb levels after a donation. There was a lot of discussion on this topic at the conference. Shouldn’t we screen for ferritin, a marker for iron stores? In the Netherlands we use ferritin testing in research only, while for instance in Scandinavia ferritin testing is already becoming part of the blood bank routine. New methods to prevent iron depletion in donors have to be proven safe and cost-efficient before these are implemented.”

Could you prevent deferral by predicting Hb?
“We aim to minimize donor deferral, as it is disappointing and time-consuming for both donor and blood bank, and an important reason of donor lapse. Donors each have personal characteristics that may determine their capability to recover from blood donations. Donors may vary in how quickly they restore Hb levels, supplement red blood cells and in their efficiency of iron uptake from food. We have recently started a study (Donor InSight (DIS)-III) in 3000 donors in which we will investigate genetic determinants in relation to declining versus stable Hb levels in repeat donors. We’ll search for a gene profile that is predictive for Hb decline so to determine the optimal donation interval for each future donor. Combining this test with a genetic array for blood types could be a cost–effective way to implement the results. In the future we hope to be able to personalize donation intervals for each donor, to prevent deferrals and keep the donor healthy.”

Donor InSight is a very large cohort study. What will you do with all the data?
“The study is generating big data that we like to share with others. We cordially invite interested researchers to contact us in order to find out if we can help them answer their research questions. Two questionnaire rounds, DIS-I in 2007-2009 (n=31,338) and DIS-II in 2012-2013 (n=34,823, of whom 22,132 also participated in DIS-I), were completed to gain more insight into characteristics of donors, their motivations and health. DIS data can (routinely) be linked to the donor database, with details on all donations, containing donor screening results including the donor health questionnaire answers, blood pressure and haemoglobin measurements. Potential research questions to be addressed with these data are not limited to blood donation-related issues. At Sanquin we have for example looked into associations between outdoor temperature and blood pressure. It is a well-known fact that mortality among elderly is higher in extremely cold and hot weather. Before each donation we measure blood pressure while the temperature is registered at a nearby weather station. We correlated the two, and found that blood pressure was lower at higher temperatures, especially at high age and the associations were significantly stronger at higher temperatures. Because the Dutch climate is temperate and the donor population is relatively healthy, we think that effects of more extreme temperatures in more vulnerable populations might be stronger.”

Are you blood donor yourself?
“Yes, I have become a donor too. I was a little anxious at first, because I usually have a rather low blood pressure. But I wanted to have a better understanding of what blood donation is about and what donors experience. And I must admit, it feels better than expected. The donor assistants did pamper me with soup and allowed me extra time to lie down. Before donations I drink half a liter of water. It has been shown that this helps to prevent vasovagal reactions after blood donation. In fact I hardly notice anything after a donation.”

“Hematopoietic stem cells walk on a narrow balance between leukemia and aging”

Marieke von Lindern is head of the department of Hematopoiesis. Her department investigates the process by which mature peripheral blood cells are formed from the hematopoietic stem cell (HSC). Important topics are aging of stem cells and the culture and maturation of various blood cell types from progenitor cells, isolated from peripheral blood.

In 2012 the Dutch Hendrikje van Andel died at the age of 115, at the time the oldest person in the world. Recently, researchers reported that at the end of her life all of her peripheral blood cells were derived from only two active HSCs. How did that come about?
“Hendrikje van Andel started life with about 1300 active HSC. During her life, her HSCs have renewed and differentiated into various lineages of the blood cell system. Almost all of Mrs van Andels HSCs were burned out at the end of her life. All those numerous replications may have caused mutations in the DNA of the stem cells. This could have led to unlimited growth, leukemia, if it weren’t for her tumor suppressor genes that regulate proliferation and differentiation. They make cells that have gained tumor characteristics to go into apoptosis or differentiation to mature cells that lack replication potential. In fact, aging protects us from having cancer. Stem cells walk on a narrow balance between the unlimited growth seen with leukemia on the one hand aging on the other hand I think Mrs van Andels stem cells have done an extremely good job in finding that balance.”

Aging is a natural occurring process. When does aging of HSCs become a problem?
“Some examples of extreme aging are found in syndromes or diseases like progeria. But what is more relevant for my department is the Myelodysplastic syndrome (MDS). Patients with MDS may develop severe anemia and frequently require blood transfusions. In some cases, the disease worsens and the patient develops low blood counts, caused by progressive bone marrow failure. There is still not much known about the syndrome and various theories about its nature exist. In some cases MDS may be caused by exposure to certain chemicals such as those in solvents that were used for paint . What we do know is that in those patients the tumor suppressor genes are upregulated, resulting in a strong pressure towards aging. If cells manage to escape from that pressure, the MDS patient may develop leukemia. As those leukemic cells have already escaped natural tumor suppression, it is particularly difficult to find effective treatment for those patients.”

Dream or reality: an injection with young blood for all elderly people?
“I think the recent reports about rejuvenating old mice by giving them young blood are highly important for aging research. To me however, prevention of aging is not a goal in itself. For some diseases that we study, however, aging may be an underlying cause. With our research on aging of HSCs we primarily aim at elderly with bone marrow failure, chronic leukemia and MDS, who are currently treated with cord blood stem cells. Therefore we work closely together with the Laboratory for Cell Therapy and the Cord blood bank here at Sanquin.”

You have received a prestigious grant for culturing red blood cells from stem cells. How does that go about?
“We start with the buffy coat, the fraction of white cells in the blood donation bag, which is usually disposed of. From this fraction we culture red cell progenitors under defined conditions. We let them first expand to a very large number of cells, and subsequently we differentiate these cells into reticulocytes. When administered to a patient the reticulocytes will further mature into erythrocytes. In theory, we are currently able to double the number of red blood cells from one donation. I think that in 1.5 to 2 years we will be ready to do the first mini blood transfusions in volunteers in order to test our culture system for safety and immunogenicity. At the same time we want to develop a more cost-effective and efficient way to culture blood cells by making stable red blood cell lines. To do so we are generating induced pluripotent stem cells (iPSs) out of blood cells, which we dedifferentiate by introducing four different transcription factors. In fact, iPSs are very similar to embryonic stem cells, but go without ethical controversies. From iPSs, which are an immortal source, we can generate any amount of red blood cell by adding a cocktail of growth factors. In addition, we’d like to study the generation and culture of erythroblasts as an alternative source for producing red blood cells.”

What kind of patients may receive cultured blood in the future?
“The reason that we want to culture blood is that for some patients it is very hard to find matching donor blood. That particularly holds for patients that frequently need blood transfusions. We regularly see patients making antibodies to donor blood, even if there is only a small difference in minor blood groups. That goes for patients with chronic anemia, MDS, but most of all for patients with sickle cell disease. Sickle cell patient are usually from African origin, their blood groups differ from the greater part of our blood bank population. The number of suitable donors for sickle cell patients is therefore limited and their blood is needed frequently. There is a great demand for matching blood for these patients. Generating red blood cells from iPSs of those rare donors could meet that demand. In addition, we could even make functional red blood cells from the sickle cell patient’s own iPSs. It appears that erythrocytes cultured from iPSs primarily express the fetal gamma globin rather than beta globin, the adult hemoglobin, which is affected in sickle cell disease. I hope that in the future we will be able to culture a bag of matching blood for each patient whenever needed.”

Getting back to Hendrikje van Andel: what is aging to you?
“To me, healthy aging is more important than aging itself. I try to enjoy life and do what is important to me now and not later in life. I am trying to live my life meaningfully. Thirty years ago we couldn’t have imagined where we are now in the field of science or society itself. I find it very difficult to think about my life thirty years from now.”

gepubliceerd op http://sanquintalks.org/hematopoietic-stem-cells-walk-on-a-narrow-balance-between-leukemia-and-aging/  juli 2014