Hematology

Hematology is the study of the blood, the cells of the blood, the proteins of the blood, the processes of the blood and even the diseases of the blood. Here we will begin our journey into the study of hematology and the diseases that occur in the blood. The blood is made up of mostly water. This makes hydration the key to life and healthy blood. When you have too little water in the blood, it gets thick and causes illness.

When we break down the major 3 components of blood, they will be Plasma which is the watery part, the Red blood cells and then the Buffy coat. We will look at each of these parts, then we will go back and look at how they are created. About 55% of the blood is the plasma. This is the watery part of the blood that the cells move through. This keeps things flowing. There are so many things inside the plasma like oxygen, carbon dioxide, vitamins, minerals, and proteins. Most of the proteins made for inside the blood come from the liver. When you talk about a protein in the blood, you can guess it comes from the liver and 90% of the time you will be right. We will cover many of them as we go. Some of these proteins are for clotting of the blood, When these proteins are not clotted, we call it plasma. When they are allowed to clot, we call it Serum. These are 2 different terms used for blood and they are important. The serum has all the clotting factors removed.

The next major part of the blood is the Red Blood Cells. They carry the oxygen that keeps our cells alive. That is their sole purpose in life. They are the most important cell in the blood. They make up about 45% of the blood by volume. We call this number the Hematocrit. This is an important number and we will dig into it deeper when we get into red blood cells. Many of the diseases of hematology will deal with defects in Red Blood cells. Red blood cells are not technically cells as they expel their DNA and nucleus as part of their development. This makes them short lived with one sole purpose in life. We will spend a lot of time on Red Blood Cells.

The Buffy Coat looks white and where it gets its name. It is made up of the many White Blood cells that make up the immune system. There are about 10 cells that are made in different amounts for the immune system, and they all fall under the buffy coat. This includes Granulocytes, Myelocytes, T cells, NK cells and B cells. The buffy coat also includes the platelets which play a role in blood clot formation. All these cells combined make up about 1% of the blood volume.

The plasma contains the hundreds of proteins that work inside the blood from clotting factors to transport proteins. They are broken down into 3 classes with alpha, beta and gamma proteins. The Gamma proteins are the Immunoglobulins also known as the antibodies. The Alpha and Beta proteins include things like cholesterol proteins among many others. Albumin makes up about 60% of all proteins in the blood. It plays a key role in regulating osmotic pressure.

All these cells that are in the blood come from a single source called the Hematopoietic Stem Cell (HSC). It is also known as the Hemocytoblast. The HSC undergoes a process called asymmetric cell replication. When the HSC replicates, it creates one exact copy of itself with a new HSC, and it also creates 1 of 2 possible progenitor cells. It will come out to about 50% of the cells created being a Myeloid progenitor and about 50% of the time the cells created will be a Lymphoid Progenitor. The Myeloid progenitor will go on to create Erythrocytes (Red Blood cells), Megakaryocytes (Platelets) along with Granulocytes and Monocytes for the immune system. The Lymphoid progenitor will create the T cells, B cells and NK cells.

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Iron Metabolism

Iron comes in 2 forms with Ferric Iron (Fe3+) and Ferrous Iron (Fe2+). Which form Iron is in at any point in the body depends on where it is currently at. We eat iron in both forms. Only the Fe2+ can enter the body in the small intestines. The stomach will convert Fe3+ into Fe2+ through chemical reactions. The saying you can use to remember this is, "Fe2 goes INTO the body and INTO the Red Blood cells". The only other source of iron in the body is recycling of old Red Blood cells by the macrophages in the spleen. The body has no natural mechanism to remove excess iron. That makes the absorption of iron a very highly regulated process. This comes into play during iron overload (Hemochromatosis). The only effective way to remove too much iron from the body is Chelation therapy. This is important in many anemias where the treatment is to give Red Blood Cells. All these new cells transferred into the patient will eventually get recycled and all that iron ends up in the body's supply causing iron overload.

Ferrous Iron enters into the Enterocytes of the Duodenum of the GI tract through a transporter called DMT1 (Divalent Metal Transporter 1). This transporter can only accept the Ferrous (Fe2+) iron which we call Heme Iron as it is the only kind of iron that can be used in Red Blood Cells. For patients who have diseases of the bowl like Celiacs or after bowel surgeries that affect the duodenum of the small bowl, they can have problems absorbing iron through food sources. They will need supplemental iron usually by muscular injection or IV.

Once the iron is inside the Enterocyte, it can be absorbed into the blood steam if necessary. The enterocytes turn over frequently. That means any iron that is not needed and absorbed by the body will be shed with the enterocytes into the waste. The movement of the iron from the enterocyte into the blood steam requires active transport by the enterocyte through a transport protein called Ferroportin. This sends the iron into the bloodstream in the form of Fe3+ Ferric iron. The iron needs to be transported in the blood by binding to a transport protein called Transferrin. That is because iron is a statically charged element. It can cause serious damage if not bound to Transferrin. The iron gets stored in the body in 2 places. The first is it is stored in the Macrophages of the Bone Marrow where it is used to create Red Blood cells. The main storage of the iron is in the liver. Liver stores of iron are regulated by a protein called Hepcidin. When the stores in the liver are full, Hepcidin will bind to the ferroportin receptor on the enterocytes and stop the intake of the iron into the body. When iron stores run low, Hepcidin will drop and allow iron to pass into the body. When iron gets stored in cells like Macrophages or the Liver, it gets bound to a protein called Ferritin. The Ferritin storage protein of iron and the Transferrin transport protein for iron are key diagnostic tools.

When iron is in need, the ferritin will drop as there is no iron to store. The Transferrin will become elevated in the blood as it is looking for any iron it can find. When iron levels are high, the ferritin will increase in an attempt to store as much of the excess as possible. The transferrin in circulation will drop as there is no need to find more iron. With this understanding, we can use ferritin and transferrin levels as diagnostic tools. We refer to them as iron studies.

Ferritin for storing iron is an acute phase reactant. This is because bacteria love and need iron to thrive. When there is an infection or inflammation, the liver increases the level of ferritin to absorb and hide all the iron from the bacteria. This becomes important in chronic diseases where there is inflammation. The body will tend to hide the iron because it thinks it has an infection. There is no defect in the body's ability to absorb or use iron. The iron is just being hidden from the body because of the inflammation. The main issue for iron in the body is iron deficiency. This is the most common anemia in the world. Most of it is nutritional anemia due to lack of food.

Iron Deficiency Anemia does apply in the US mostly around a few key areas. The first in Vegan and Vegetarian people who don't pay attention to their nutrition as most heme iron comes from meats. Since Iron is so important to Red Blood cells, iron deficiency anemias will occur where heavy cell creation is being done like pregnancy and early child development. This is why we use prenatal supplements to give extra iron and vitamins. Iron's role in the body will be to create the Hemoglobin for the red blood cells. That is because the iron molecule is charged, and it binds oxygen very well. We will look deeper at Iron Deficiency Anemia later.

There is a series of tests for the iron in the body called iron studies. They include the level of free iron in the circulation which is low because it can be harmful called serum iron. It includes the ferritin levels which can be a sign of iron sequestration due to chronic disease. The final test is the Total Iron Binding Capacity (TIBC). This measures that amount of the transferrin that is in circulation by measuring the number of binding capacity. The last number you may see in these studies is the saturation. Iron saturation can tell you how much of the total capacity of the Transferrin is actually binding to iron. This is usually represented as a percentage. This is usually 20% to 50%. High levels could indicate the body is in need of iron.

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Red blood Cells

The purpose of Red Blood Cells (RBCs) is to pick up, transport and deliver oxygen. They are made up of about 270 million hemoglobin (Hb). Hemoglobin is a complex protein structure we will look at after Red Blood Cells. The RBCs pick up oxygen in the lungs, and they travel through the blood circulation until they reach the small blood vessels of the tissue where the oxygen will be released. This provides the necessary oxygen to the tissue cells for survival.

The oxygen levels in the blood are sensed by the Kidneys. When O2 levels are low, the kidneys will release the growth factor Erythropoietin (EPO). This will travel to the bone marrow and act upon the Hematopoietic Stem Cells (HSC). This brings us to our first possible disease that leads to anemia. This is anemia of chronic kidney disease. Since the kidney is critical for regulating RBC development by sensing O2, it can become dysregulated in disease of the kidney or even in kidney cancers.

The Erythropoietin (EPO) will enter the bone marrow and stimulate the HSC to make more Myeloid progenitor cells. It will also act on the Myeloid progenitor cell to create more Erythroblasts. These are special cells that make large quantities of Red Blood Cells (RBCs). The body has about 4 to 6 million RBCs per microliter of blood that means we have about 25 trillion RBCs at any time with 5 liters of blood in the average person. We manufacture about 2 million RBCs per second.

The RBC lives about 120 days. When it is first created, it has a biconcave shape. This makes the RBC very flexible. As the RBC ages, it will become rigid. There is a slit in the spleen where the spleen macrophages line the blood vessels. Each RBC will have to squeeze through this slit. The older RBCs become rigid and will not make it through. Then the macrophages will ingest them and break them down. This is a natural process to remove old RBCs and recycle their iron to make new ones. The Spleen macrophages are capable of removing about 5 million old RBCs per second. All the iron is recycled for use in new RBCs while the rest becomes waste products like Bilirubin.

The new Red Blood Cells will be created in the Bone Marrow by the Erythroblasts. The early stage of the Red Blood cell will be a large cell with a nucleus and DNA. They begin to make Hemoglobin. They will pack about 270 million Hb into every RBC. As the RBC develops it will actually shrink in size. The last thing the RBC will do before it leaves the bone marrow will be to eject its nucleus and DNA. Then it will be released into circulation as a Reticulocyte. The Reticulocyte will take a few days to fully mature into a Red Blood cell.

This brings us to another meaningful clinical test. The Reticulocyte count is about 1% to 3% of the circulating RBC population. If this number is lower than 1%, you might have a production problem with RBCs. If you have more than 3% reticulocyte count, then you might have bleeding or a destruction issue. After a few days, the reticulocytes will become fully mature. They will be about 7 to 8 microns in diameter and take on the biconcave disc shape.

The RBC retains its shape through structural proteins like spectrin and ankyrin. This brings us to our first possible defect in the actual RBC. There are mutations in these structural proteins which lead to misshapen RBCs. This can lead to Spherocytosis and Elliptocytosis. These misshapen RBCs won't pass through the slit in the spleen. Then it will get destroyed by the spleen macrophages. This leads to a destruction anemia where the RBCs are destroyed due to being misshapen.

Erythropoietin is considered an cytokine and growth factor for Red Blood cells. It binds to the JAK-STAT pathway receptor. There are genetic mutations in this JAK receptor that will lead to an "always on" effect like the JAK V617F mutation. This leads to an overproduction disease like Polycythemia Vera, Essential Thrombocythemia and Myelofibrosis. These are called Myeloproliferative Disorders. We will look at these later in Hematology Oncology.

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Hemoglobin

Hemoglobin is made up of Heme and Globin. The structure of Hemoglobin is made up of iron (Fe2+), Protoporphyrin, and a group of 4 proteins called Globins. The Globins are made up from 2 genes called the Alpha Globin gene and the Beta Globin gene. Two of each of these globin chains will come together to form a 4 leaf clover like structure. It is made from 2 alpha globins and 2 beta globins. At the center of the 4 leaf clover-like Hemoglobin structure is a protein called Protoporphyrin IX. This kind of looks like a snowflake in its structure. It is synthesized from a long pathway of many enzymes that creates the Protoporphyrin protein. In the very center of the Protoporphyrin will bind a molecule of iron. The Iron plus the Protoporphyrin is the Heme. The 4 globin proteins that make the 4 leaf clover structure are the Globins. Together it forms hemoglobin.

Here comes our first set of diseases in Red Blood Cells. There is a group of diseases called Porphyria that stem from defects in the enzymes which lead to a failure to make the protoporphyrin molecule. The intermediate steps of protoporphyrin synthesis create intermediates that are toxic. When the next step fails due to a missing enzyme, the build up of these toxic intermediates occurs. This leads to Porphyria. There are a few types of this disorder that attack the nerves and skin.

Once the protoporphyrin is formed properly it will bind a single molecule of iron in the center. The only thing that can go wrong with iron is a lack of it. This is probably one of the largest anemias in the world. The lack of iron leads to Iron Deficiency Anemia and the major causes are malnutrition, pregnancy and child growth.

The Globin is made up of 4 actual globin proteins which come together to form a 4 leaf clover like structure. Now we need to look at these 4 globin proteins as they come in 2 proteins and there are diseases that stem from them. When the Embryo is in early development, the Red Blood Cells will make Hemoglobin with 2 Zeta and 2 Epsilon globin proteins. This is called Embryonic Hemoglobin (HbE). As the baby develops into a fetus, it will begin to produce hemoglobin using 2 Alpha and 2 Gamma globin. This will be called Fetal Hemoglobin (HbF). After the baby is born, the hemoglobin will start being produced using 2 Alpha and 2 Beta globin proteins. This is called Adult Hemoglobin or HbA. The famous gene BCL11A regulates the transfer from Fetal Hb to Adult Hb. From the time the baby is born until about 2 years of age, the process of transfer from Fetal to Adult Hb will take place. There are actually 2 kinds of Adult Hb. The first is the common hemoglobin which takes 2 alpha and 2 beta globin proteins. This is called HbA1. The other is another adult Hb that is made up of 2 alpha and 2 delta globin proteins. This is called Adult Hb type 2 or HbA2. The blood consists of about 95% to 98% Adult HbA1 and 1% to 3% Adult HbA2. In the average adult Fetal Hb will be made at less than 1%.

The first disease from Globin genes comes from the Alpha globin genes. There are 4 genes for these proteins with 2 coming from mom and 2 coming from dad. Depending on how many of the 4 genes are lost will determine what level of Alpha Thalassemia the patient will have. The next disease comes from the Beta Globin genes. There are only 2 of these genes. You get 1 from mom and 1 from dad. These mutations can vary in the level of Beta globin that is produced from the full amount to absolutely none. The low production or loss of Beta globin leads to Beta Thalassemia. Patients with both genes making no beta globin are the most severe form of Beta Thalassemia. This is often called Beta Thalassemia major.

The last disease that originates from the production of Hemoglobin is Sickle Cell Disease. This comes from a single point mutation in the Beta Globin. This results in a fully functional Beta Globin protein under most conditions. These mutated forms of Hemoglobin Beta are named Hemoglobin S (HbS) for Sickling. This single point mutation causes the hemoglobin to bind together into long chains when the oxygen level gets too low. This polymerization of the HbS causes the Red Blood Cell to take on a Boomerang shape. These sickle shaped Red Blood Cells can block vessels and cause potentially life threatening blockages. This can lead to intense pain and even more serious events like a heart attack, pulmonary embolism or stroke. These odd shaped cells will often get destroyed in the spleen which leads to anemia.

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Lab Tests

Here we will look at the common values used in checking for anemia. All of them come from the typical Complete Blood Count (CBC) blood test. Most doctors do one of these annually to ensure all things are well. The values I give will be a range. These are the typical values in the literature. They may vary some between different labs. Some labs use different units of measure. I have seen some labs use the standard units here while others use all things adjusted to liters. The ranges will be right, but you might see 14/dl Hb at one lab and 140/liter at another lab. Just watch the units being used and adjust.

The first series of lab values will be Hematocrit, Hemoglobin and Red Blood Cell counts. When these values are low, it is a sign there might be anemia of some kind. The physical symptoms of anemia are Pale, Tired and possibly shortness of breath. The Hematocrit measures the level of RBCs as part of the total blood volume. This tends to be in the 35% and 50% range. Women will tend to be closer to the lower half of the range and men the upper half. The average is considered 45%. A low hematocrit is called anemia while a high hematocrit is called polycythemia. This can be caused by altitude or elevation. People who live at high elevations tend to have higher hematocrit levels. Dehydration can also cause a high hematocrit level. Anemia is a disease of low Red Blood Cell counts and polycythemia is a disease of high Red Blood Cell counts.

The next up is Hemoglobin (Hb). This is a measure of the amount of total Hemoglobin. This will be in the 12.5 to 16 g/dl level. Women tend to be toward the lower end of the range and men near the upper end of the range. Some labs will express this as grams per liter like 125 g/liter to 160 g/liter. Hemoglobin carries the oxygen and low level will be a sign of anemia.

The last one is the Red Blood Cell count. This will be in the range of 4 million to 6 million RBCs per microliter of blood. Again women tend to be in the lower range while men will tend to be near the upper end of the range. I have seen tests where they express this in a exponential format using a per liter format which will be like 3.5 - 6 * 10E12. These lab values plus any symptoms from the patient would help point toward anemia, but there are dozens of possible causes for anemia. It can be blood loss, autoimmune, genetic defect or even hemolytic. It will require more labs.

The next set of values will be the Mean Corpuscular Volume (MCV). This is the measure of the average size of the Red Blood cells. This comes in a standard range of 80 to 100 fl. If the value is in the normal range, it can indicate one of the normocytic anemias. Anything below 80 fl will be considered a microcytic anemia and anything over 100 fl will be considered a macrocytic anemia.

The next important count is the Reticulocyte count. This measures the amount of immature red blood cells in circulation. The reticulocytes should range between 1% and 3%. If this is below 1%, it can be a sign of an underproduction anemia. With underproduction anemia, something is wrong or missing which leads to blast cells not developing enough RBCs. There are just not enough RBCs able to be made. When the reticulocyte count is over 3%, then it could point to a destruction anemia or blood loss. This means the blood is making tons of new red blood cells trying to make up for the loss. This is where the next set of lab tests can come in.

The next 3 tests are called the tests of Hemolysis. They measure if Red Blood cells are being destroyed or broken down. This is a key set of labs if the concern is a destruction anemia vs something like blood loss due to a GI bleed. The first is Lactate Dehydrogenase (LDH) which is an enzyme used in cell metabolism. When the LDH level is high, it is a sign of high cell turnover suggesting cell destruction. The next is Unconjugated Bilirubin which is a molecule that is created through cell break down. High levels of unconjugated bilirubin is a sign that cells are being destroyed. The last one is Haptoglobin. We already know that iron when left in the blood without a transport protein can cause big damage. Haptoglobin is a protein in the blood that is there to absorb any hemoglobin released due to cell destruction. When red blood cells are destroyed in the blood vessels the haptoglobin level will drop. This is another sign of red cell destruction.

These series of tests will help inform the doctor about whether or not anemia exists and what type of anemia is present. There is also a set of iron studies that report on serum iron, TIBC and Ferritin. These can be used if you think that the problem might be iron related.

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Hemostasis

I will prefix this section because I have not yet covered Platelets or Coagulation. This will make references to those systems, but I will try to keep it more of a general overview. Hemo means blood and stasis means equilibrium. This is the understanding that blood in circulation is kept in the liquid form. When injury occurs or blood is removed from the circulation, it turns into a solid clot. The star of hemostasis is the Endothelial cells that line the blood vessel walls. These cells are biologically active cells for cardiovascular control, blood clotting, and immunology.

In a healthy circulation, the Endothelial cell has 3 roles. The first is to promote Vasodilation. To do this the Endothelial cells synthesize Nitric Oxide and Prostacyclin (PGI2). These 2 mediators will act upon the smooth muscles that line the vessel walls. This causes them to relax and the vessel dilates. This keeps the blood vessel at normal size. In infections and injuries, the vessel can dilate even more to promote more blood flow. This causes swelling. In and bleeding injury, the endothelial cell can promote constriction which slows blood flow and prevents blood loss. When injury comes, the loss of the endothelial cells by injury or their damage will cause the loss of these dilators like Nitric Oxide and PGI2. The activated platelets will produce vasoconstricting mediators like Thromboxane A2 and Serotonin that stimulate the vessels to constrict to slow blood flow. This places a balance between healthy endothelial cells promoting vasodilation while activated platelets will promote vasoconstriction around the area of injury.

The next role of healthy endothelial cells is to prevent the activation and aggregation of platelets. Platelets are like small packets of glue that clump up and stick to each other when they are activated. That is how they cause a clot and block blood loss. When all things are healthy in the circulation, the endothelial cells will release mediators that keep the platelets from activation. The platelet needs ADP to become active. The Endothelial cells will release phosphatases that degrade the ADP into AMP and prevent platelet activation. When the endothelial cells are lost or damaged in injury, the loss of this inhibitory mechanism will allow localized platelet activation. This is how the blood circulation regulates blood in liquid form but allows localized platelet activation in the presence of injury. When you see plaques build up in cardiovascular disease, they disrupt this process and you get localized platelets sticking inside the vessel.

The last job of the healthy endothelial cell is to block coagulation. This is a group of about a dozen protein factors in the blood that create a mesh like web over a platelet clot to make it stronger. These factors are in circulation in an inactive form. They become activated in injury. This leads to coagulation which happens in the plasma of the blood. Coagulation can happen completely independent of any other cells or mechanisms. This is why blood clots in a test tube and separates plasma from serum. The healthy endothelial cells will express multiple proteins on their surface that block coagulation factors that are active anywhere in their area. They express Heparin Sulfate which works with Antithrombin to deactivate thrombin. They express thrombomodulin which binds the famous Protein C which in turn deactivates factor V and VIII. The last one is the healthy endothelial cells release Tissue Plasminogen Activator (TPA).

These 3 roles of endothelial cells of controlling vasodilation, inhibiting platelets and inhibiting coagulation is how the blood stays in liquid form when the endothelial cells are healthy. Once the endothelial cells are lost by injury or damage, those mechanisms are lost and localized clotting can occur. The clotting process is broken down into 2 stages even though they often occur simultaneously. Primary Hemostasis happens with platelets and it is the formation of the platelet plug that stops the blood loss. Then Secondary Hemostasis will happen during the process of coagulation. This reinforces the platelet plug with a meshwork of Fibrinogen fibers.

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Arachidonic Acid Pathway

So what is Arachidonic Acid? It comes from the lipid membranes of the cells. When most people think of inflammation, they think of immune cells and cytokines like TNF-a, IL-1b, C3a, C5a, or INF. These are all cytokines released by the immune process to promote inflammation. But not all damage is detectable by the immune cells. Sometimes it is an injury like a burn, cut or even chemical damage. This is where the tissue damage pathway that is Arachidonic Acid comes into effect. There is coordination and cooperation between the inflammation of the immune cells and the inflammation of arachidonic acid. Many people who study immunology miss this critical part about tissue damage inflammation.

So how does this work? It all starts with the phospholipids that make up the membranes of cells. These have a phosphate group linked to 2 long chain fatty acids. We get them from the foods we eat. They make up the phospholipid bilayer of the cell membrane. When the cell takes damage like from injury or from toxin released from pathogens like bacteria, the phosphates break loose. This gets converted into Arachidonic acid by an enzyme. The conversion of fatty acids from lipid membranes into Arachidonic Acid is done by an enzyme called Phospholipase A2 (PLA2). This converts these fatty acids into Arachidonic Acid. This PLA2 enzyme is inhibited by steroids like Prednisone. This is how steroids block the immune response and inflammation. Once the Arachidonic Acid is formed, it will be converted by 2 other enzymes which will take it down 2 different pathways.

One of the 2 enzymes is called Lipoxygenase which is called the LOX pathway. This creates Leukotrienes. Most of these play a role in smooth muscle in the lungs. This leads to bronchoconstriction. This plays a role in many disorders of the lung and anaphylaxis. Some of the many drugs that work in the lungs will affect the formation of Leukotrienes by the LOX enzyme. The only Leukotriene that doesn't work in the lungs is Leukotriene B4 (LB4). This is a chemotactic agent that recruits immune cells to the site of the damage. This plays a role in inflammation and the recruitment of neutrophils which clean up bacterial infections. Where there is damage, there is usually infection by bacteria.

The other enzyme is Cyclooxygenase which is called the COX pathway. This creates Prostaglandins. This is where things get very interesting. The COX enzymes can be blocked by over the counter pain medications like NSAIDs. This leads us to some of the benefits and side effects of these over the counter pain meds. The first is the formation of Prostaglandin E2 (PGE2) which leads to pain and fever. Prostaglandin D2 and F2 (PGD2 and PGF2) actually work in the stomach to promote healthy protection from the stomach acid in the lining. This is why over-the-counter NSAIDs can lead to stomach upset and sometimes bleeding and damage with chronic use. The last 2 major Prostaglandins take the PGH2 and synthesize the Prostacyclin (PGI2) or Thromboxane (TXA2). If you recall our intro to hemostasis, these are made by endothelial cells to regulate vasodilation and vasoconstriction. When the endothelial cells are healthy, they convert the prostaglandins into PGI2 which promotes healthy vasodilation. When platelets are active, they will convert the prostaglandins into TXA2 which promotes vasoconstriction to slow blood loss. This makes the Arachidonic Acid pathway critical in both healthy blood flow and inflammation.

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Endothelial Cells

This will cover some of the early responses to inflammation and hemostasis with the preformed elements contained in Weibel-Palade Bodies. The initial response to injury often contains both hemostasis to control bleeding and inflammation to control infection. These two things tend to come together. Any injury that will puncture a vessel will most likely bring with it bacteria. For cells to respond to this immediate damage, it takes hours. The cells have to turn on the right genes, copy them into messenger RNA and then make the proteins. If we had to rely on this process to survive, there would be no humans.

The initial responses to inflammation and hemostasis comes from preformed elements contained in granules in various cells. For example, the Mast cells of the immune system that line the skin will release histamine from granules to promote inflammation. Platelets contain granules which contain clotting factors. The Endothelial cells also contain granules called the Weibel-Palade bodies. This contains both P selectin and Von Willebrand Factor (VWF). The P selecting works with immune cells to traffic them into the tissue. The VWF will be carried by the blood that is escaping the vessel through injury. It will coat and bind to the collagen and extracellular matrix providing a surface for platelets to stick to. The Endothelial cell itself is a very active cell that makes up the blood vessel walls. They play a key role in both inflammation and hemostasis. They respond to cytokines from inflammation.

These are signals like TNFa and IL-1b release from immune cells, it can be C3a and C5a from the complement system, it can be histamine from mast cells, and it can be prostaglandins and leukotrienes from the Arachidonic Acid pathway. The Endothelial cells have receptors for these cytokines. They will respond by promoting Vasodilation in inflammation, and will even shrink to increase vascular permeability. The increased flow out of the vessel helps get immune cells and other factors into the tissue to help with clearing infection or repairing damage. The Endothelial cells of the blood vessel play a major role in helping traffic cells into tissues. They will respond to inflammation with release of the Weibel-Palade bodies with P selectin. This will be the primary response. After a few hours, they will begin to produce and express E selectin. This is the secondary response. The secondary response will come from the inflammatory mediators like TNFa and Il-1.

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Platelets

Platelets are called Thrombocytes for their technical name. You will see this term used a lot in literature. I will use the common name platelet. They are not technically cells as they do not contain any nucleus or ribosomes. They are created from Megakaryocytes in the bone marrow. Each Megakaryocyte makes about 2,000 platelets each. They are made as part of the Myeloid Progenitor cell line that comes from the stem cells in the bone marrow. The platelets are just membranes with receptors and packed with granules with preformed elements in them. The megakaryocyte pinches off these little fragments of cells. They are then released into circulation. The size of the platelet is about 2 to 3 microns. That makes them about 1/3 the size of the Red Blood cells. The normal amount of Platelets is between 150,000 and 450,000 per microliter of blood. If the platelet count is low, it is called thrombocytopenia. When the platelet count is too high, it is called thrombocythemia.

The platelets in normal circulation will be completely inactive. The healthy endothelial cells will inhibit the platelets. The purpose of the platelets are to be like sticky little packets of glue that block punctures in the blood vessels when activated. They stick to the VWF released by the Endothelial cells. They can also bind to the collagen or extracellular matrix. This causes them to activate and recruit more platelets to build a dam that blocks blood loss. Platelets have several receptors on their surface. The first is the ADP receptor which activates the platelet. If you recall, healthy endothelial cells will break down ADP to prevent platelet activation. The other key receptor is the GP1b receptor which binds to the VWF that is released by the endothelial cells. The VWF coats the wound by binding to collagen and extracellular matrix. This gives the platelet a place to bind with the GP1b receptor. This is Platelet Adhesion. This is the process of a platelet sticking to a non platelet surface. Then there is a GPIIa/GPIIIb receptor. It is called the 2a/3b receptor. This allows activated platelets to stick to other platelets using Fibrinogen. This causes Platelet Aggregation. This is the term for platelets sticking to other platelets. The last part of the Platelet is the granules. They have Alpha and Dense granules.

The dense granule has 3 key elements in them. The first is Serotonin. This works as a vasoconstrictor. The platelet takes prostaglandins from the Arachidonic Acid pathway and will use them to synthesize Thromboxane A2. With these two Vasoconstrictors, the platelet controls vasoconstriction. The dense granule has ADP. This will be released and bind to other nearby platelets activating them. The last is Calcium (Ca2+). This plays a role in coagulation so I will cover that later. The Alpha granules of the platelet have a few key coagulation factors like Fibrinogen, inactivated Factor VIII and V. We will cover them in coagulation.

Platelets live about 8 to 10 days. They are inhibited by ADP inhibitors like Clopidogrel. Aspirin is also an irreversible platelet inhibitor. Aspirin is a COX inhibitor of the Arachidonic Acid pathway. This inhibits platelets by blocking its ability to produce Thromboxane A2. The only way the Aspirin wears off is by waiting a few days until the platelets that are inhibited by Aspirin are removed and replaced with new platelets. This is why Aspirin is used as a platelet medication in cardiovascular disease. Often in cardiovascular disease, the plaque buildup in the blood vessels will block the anti platelet effects of endothelial cells. This leads to platelet activation and clotting in the blood vessels. Using aspirin can help with preventing activation and sticking of platelets. This also leads to the potential bad side effect of Aspirin. Aspirin blocks the ability of platelets to activate. This means you have a higher risk of bleeding when taking Aspirin.

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Coagulation

Coagulation is a process governed by about a dozen proteins that work together to take soluble Fibrinogen and turn it into activated Fibrin monomers. These will connect together into long strands that bind to the platelet plug and strengthen it. The first thing you will notice is coagulation comes in two pathways called the intrinsic and extrinsic. Most of these proteins are numbered using Roman numerals. They circulate in the blood as inactive enzymes. When one becomes active it will activate the next. There is one key component to activation of these proteins. They require Vitamin K to activate. That is how the anticoagulant Warfarin works. It blocks vitamin K which prevents activation of coagulation factors.

The intrinsic pathway starts with Factor XII. It is called intrinsic because the doctors who first worked with blood realized that it clotted when removed and put into a test tube. They figured there must be something Intrinsic to the blood itself that made it clot when removed. We know today that this factor is Factor XII. We are still not sure exactly how Factor XII stays inactive in the circulation of the blood, but activates whenever it is removed. The Factor XII also plays another role in the Kallikrein-Kinin system. I will get into that later. When factor XII is activated, it will activate Factor XI which will in turn activate Factor IX. This will then activate Factor X. Factor X is a key factor that begins the common pathway from which all the rest takes place.

The Extrinsic pathway starts with tissue damage. This is the most common pathway by which coagulation begins. It starts with injury and it activates Factor VII. Factor VII becomes activated when it comes into contact with Tissue Factor (TF). This is found in the tissue surrounding the vessels. When Factor VII comes into contact with TF, it will become active and directly activate Factor X. At this point, it has reached the common pathway by activating factor X. Everything beyond this point is shared by both pathways.

Factor X will activate and begin to convert Prothrombin into active Thrombin. Thrombin is the most powerful part of the coagulation cascade. It doesn't matter if Factor X becomes activated by the extrinsic or intrinsic pathways, it will convert Prothrombin into Thrombin. That is where it gets exciting. Thrombin has many roles. The first is it will go back and activate more Factor XI. This allows for the activation of the intrinsic pathway if it has not already been engaged. The Thrombin will also activate the Factor VIII that was released by platelets. This activated Factor VIII expressed as VIIIa will combine with activated Factor IX expressed as IXa. Notice the small a on the end of the factor once it has been activated. Remember the Ca2+ that was released by the granules? This will bring together the VIIIa and IXa and bind them together on the surface of the platelet. That creates what is called the Tenase. This new enzyme will activate hundreds of times more Factor X. This amplifies the coagulation response.

The Thrombin will also activate the Factor V that was released by the granules. This Va will combine then with the activated factor Xa and create another enzyme complex on the platelet surface. This enzyme will be called the Prothrombinase complex. This amplifies the level of conversion of Prothrombin into Thrombin. This helps amplify the process. This binding of the Prothrombinase complex is also bound together by the Ca2+. This works like a blue between the 2 active enzymes to bind them together.

Thrombin will also activate the Fibrinogen into Fibrin monomers. This is the final goal of the coagulation cascade. These fibers will bind to the platelets Gp2b/3a receptors and make a web-like adhesion across the platelet plug reinforcing it.

The final role of the Thrombin will be to activate Factor XIII. This acts as an interlacer. It binds the strands of Fibrin together. This helps to strengthen the platelet plug. The entire process is designed to form a clot.

When the clot forms outside of the blood vessels in the tissue, we call this a clot. When they form inside the vessels, we call it a thrombus. This is what happens in cardiovascular disease where the endothelial cells are damaged or blocked by plaques. This allows for clots to form inside the blood vessels. These are called a thrombus. When the thrombus breaks loose into the circulation, it is called an embolus. These are dangerous and can lead to a Stroke, Heart Attack or Pulmonary Embolism. These can be deadly.

After the tissue heals and the endothelial cells are returned to health, they will begin to release Tissue Plasminogen Activator again. This will convert a protein called Plasminogen into plasmin. This will break down the Fibrinogen to dissolve the clot. This leaves Fibrinogen Degradation Products (FDP). There is a blood test called D-dimer. This measures the levels of these FDP products in the blood. A high D-dimer level indicates clotting and breaking down of clots.

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Hemostasis Walkthrough

Primary Hemostasis is a process of the platelets forming the platelet plug. Secondary Hemostasis is the coagulation cascade creating the Fibrin mesh that reinforces the platelet plug. These processes are taught separately to make them easier to understand, but in reality, they would both occur at the same time. Let us do a walkthrough with an example of a nail puncturing the skin.

The damaged Endothelial cells will release the contents of their granules, the Weibel-Palade bodies, which contain Von Willebrand Factor (VWF). The VWF will coat the surrounding collagen and extracellular matrix providing a surface for platelets to stick. The GP1b receptors on the platelets will bind to the VWF as they are pushed out the circulation with the blood flow. This is called platelet Adhesion as they stick to a non platelet surface. They will become active and release their granules. The activated platelets will synthesize the prostaglandins from the Arachidonic Acid pathway from the breaking down cell membranes to produce Thromboxane A2 along with releasing Serotonin from their granules. These 2 factors will promote vasoconstriction. The ADP released by the platelets will go on to bind to the receptors on other platelets nearby and activate them. They will begin to stick to the other platelets using their GP2b/3a receptor and Fibrinogen released from the platelets granules. This begins platelet Aggregation which is platelets sticking to other platelets. The platelets will continue to aggregate and release granules to activate more platelets until a platelet plug is formed. This is the process of Primary Hemostasis.

The Factor VII coagulation factor will be forced out of the blood and into the tissue which will cause it to be activated by the Tissue Factor (TF). This will then activate Factor X which will convert Prothrombin into Thrombin. Once Thrombin is activated it goes back and makes the amplification loop by activating Factor XI which activates Factor IX. The Thrombin will also activate Factor VIII released by the platelets. The active Factor VIIIa and IXa will come together with Ca2+ and create the Tenase. The Tenase complex will activate 100x more Factor X. The Thrombin will also activate Factor V which was released from the platelets. The active Factor Xa and Va will combine with Ca2+ to create the Prothrombinase complex on the platelet surface. The Prothrombinase complex will activate large amounts of Prothrombin into thrombin. This will convert the Fibrinogen in the blood into active Fibrin strands that coat the platelet plug and bind it together. Thrombin will also activate Factor XIII which will interlace the fibrin strands into a mesh like web. This will reinforce the platelet plug and create a solid clot. This is the process of Secondary Hemostasis.

Then the Endothelial cells will be replaced by mitosis and a healthy Endothelial lining will be re-established. This will begin to release Tissue Plasminogen Activator (TPA). This will convert the Plasminogen in the blood into active plasmin. This begins to break down the Fibrin strands into Fibrin Degradation Product (FDP). The clot will be dissolved and the healthy endothelial tissue will be new. Hemostasis is restored.

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Hemostasis Labs

Here we will look at the tests for healthy hemostasis with platelet count, D-dimer, Prothrombin Time (PT), Partial Thromboplastin Time (PTT) and International Normalized Ratio (INR). The first lab test for measuring the health of a patient's platelet and coagulation factors is the platelet count. If you remember, the Red Blood Cells were 5 million per microliter. The platelet normal range is 150,000 to 450,000 per microliter. You can sometimes see this represented from some labs using scientific notation with per liter measured like 150 to 450 x 10^9/ Liter. If there are too few platelets, this is Thrombocytopenia. A low platelet count, below 150,000, will lead to increased bleeding. A platelet count over 450,000 could lead to increased clotting. This is called Thrombocythemia. The platelet count can be found on the Complete Blood Count (CBC) lab test.

The next test is the D-dimer level. This will measure those Fibrin Degradation Products (FDP). This is represented in grams per liter. The normal range is less than .5 grams/liter. A high D-dimer level is an indication that clotting is happening.

The next set of tests are for the Coagulation cascade. Here we have 2 that measure each of the Extrinsic and Intrinsic Pathways of coagulation. An elevated value in one of these tests but not the other can be a sign there is an issue in that specific pathway. The Prothrombin Time (PT) will measure the amount of time it takes for the Extrinsic pathway to form a clot. The normal time is 10 to 13 seconds. An elevated PT time could indicate there is an issue in the Extrinsic pathway for coagulation. The other test is the Partial Thromboplastin Time (PTT). This measures the Intrinsic pathway. The normal time for PTT is 25 to 35 seconds. Elevated times here could suggest something is wrong in the Intrinsic Pathway. These 2 tests can be used to help isolate Hemophilia. Hemophilia affects the intrinsic pathway of coagulation. A prolonged PTT while normal PT time could be a hint at Hemophilia which affects Factor IX or VIII to form the Tenase complex.

Another test is the International Normalized Ratio. This takes the Prothrombin Time (PT) and normalizes it compared to what a normal PT time should be. This is measured in the normal range of .8 to 1.1. This test has become more widely used than PT anymore.

There can be reasons when a doctor wants to prevent blood clotting. This could be in cardiovascular disease where the Endothelial cells are damaged. This causes plaques and allows for intravascular clotting. In this situation, the platelet is the problem. This is where an anti platelet drug will work. This can be an ADP inhibitor which will stop activation of platelets. There are several ADP inhibitors on the market for this job. Aspirin is a modest platelet inhibitor which can be used in place of ADP inhibitors or in combination with other therapies. Other platelet inhibitors can be things that block GP2a/3b receptors to prevent platelet aggregation.

In the case of some diseases or in the hospital, they might want to promote Anticoagulation. These therapies block coagulation with things like Thrombin or Xa inhibitors. Warfarin is a Vitamin K inhibitor which blocks the activation of most coagulation factors all at once. The problem with Warfarin is it is very unpredictable and constant monitoring of the INR level has to be done. Heparin is a powerful anticoagulant used in the hospitals to prevent clotting in IVs and after surgery. It activates Antithrombin which blocks all thrombin that is activated. There are other Anticoagulants with the Xa (activated Factor X) inhibitors. They work really well without the constant monitoring of Warfarin. Now that there is a reversal agent on the market, the Xa inhibitors are getting much more use. They inhibit the activated Factor X. When a doctor wants to promote anticoagulation for treatment, they will use the INR test.

A normal INR is 1. When doing "blood thinners" is what they call anticoagulant treatments, they will keep the INR in the 2 to 3 range. This will significantly increase bleeding time and increase the risk of serious bleeding. The one critical note when using Warfarin (if anyone uses it anymore) is that it initially increases clotting for the first few days. This is because Warfarin affects Vitamin K. This also plays a role in Factor C which is part of the endothelial cells preventing clotting in healthy circulation. When starting Warfarin treatment, it will inhibit Factor C which can cause clotting at first. They usually use Heparin in combination for the first few days. This is important in early treatment with Warfarin. It is one of the reasons why it is getting used less and less now that other anticoagulation drugs are available like Xa inhibitors. They just block Xa which shuts down coagulation.

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Defects of Hemostasis

The first disease of hemostasis we will look at is Von Willebrand Disease. This stems from a genetic disorder inherited in an Autosomal Recessive pattern. This leads to no or little functional Von Willebrand Factor being produced. It affects about 17 in 100,000 people. This is typically a mild disease as most patients are unaware they even have it. If you recall, VWF will coat the collagen and extracellular matrix so platelets can stick and activate. Platelets can stick directly to the surfaces with other receptors. This disease slows the process of the platelet plug but it is still functional. This increases bleeding times and brings with it increased risk of bleeding events. The range of symptoms can be mild to severe depending on the level of VWF being produced.

The next set of disorders comes from the coagulation cascade with Hemophilia. There is Hemophilia A, Hemophilia B and Hemophilia C. There are about 15,000 Hemophilia A patients in the US. This is a genetic disorder in the Factor VIII production. These patients have little to no factor VIII and have problems with coagulation. Hemophilia B has about 5,000 patients in the US that come from defects in the gene for Factor IX. This leads to problems with clotting. Hemophilia C is a very mild disorder that most patients don't even realize they have. This is a defect in the Factor XI gene that helps activate Factor IX. These patients have increased bleeding times which can be problematic during dental or surgical procedures.

The last set of disorders comes from platelets. There are a few that are actual genetic defects in platelets. There is Glanzmann Thrombasthenia which is a defect in the GP2b/3a receptor which allows platelets to bind together. The other is Bernard Soulier Syndrome. This is a genetic defect in the GP1b receptor which allows the platelets to attach to the VWF. Both these defects in platelets affect about 1 per 1 million people. They are extremely rare and lead to higher risk of bleeding.

Another defect is Factor C deficiency. This one leads to the higher risk of clotting. If you recall, Factor C will bind to Thrombomodulin on the Endothelial cells and it deactivates any Factor VIIIa or Va. This is important to keeping healthy circulation. This can be inherited in an autosomal dominant pattern from mild to severe levels of Factor C deficiency. The severe form is extremely rare at about 1 in 4 million people.

The last cause can be immune related. We talked in anemia how low RBC counts could be caused by cells being destroyed by antibodies or complement. In the same way the immune system can turn on platelets. This leads to platelet destruction and thrombocytopenia. This is often transitory and related to infections. The platelets get destroyed as part of the response to the infection. The most common treatment will be steroids to suppress the immune system.

The final group of disorders are the Thrombotic Microangiopathic (TMA) disorders. They tend to affect both RBCs and Platelets. They tend to have Hemolytic Anemia, Thrombocytopenia and clots in the small vessels. This includes diseases like aHUS or TTP. These are both disorders of clotting. aHUS is a disorder of one of the factors H or I that prevent complement activation. This leads to hyperactivation which causes the complement to attack both RBCs and Platelets. It can also cause clotting.

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Contact Activation Pathway

Here we will look at the Kallikrein and Kinin pathway. This plays a role in inflammation, coagulation and pain. The endothelial cells work as biologically active cells that are the gatekeepers between the circulation and the tissue. To cover this, we have to cover a bit of inflammation. Inflammation starts with tissue damage or infection. The tissue damage will activate the Arachidonic Acid pathway with Prostaglandins and Leukotrienes. They will act upon the Endothelial cells to promote inflammation. A pathogen in the tissue can activate inflammation through Histamine from Mast Cells or TNFa and IL-1b released from Sentry cells. All these inflammatory mediators will act on the endothelial cells to promote inflammation. This has 2 effects. It causes the endothelial cells to release vasodilators like nitric oxide or prostacyclin (PGI2). They also promote the endothelial cells to contract and open gaps between them. This increases vascular permeability. This leads to the fluid of the blood, but not the cells, to flow out of the vessels and into the tissue. This includes the water of the plasma and the proteins in the plasma. This includes complement proteins, coagulation factors and proteins of the contact activation system.

There are 3 proteins that circulate in the blood that work in the Contact Activation System. They are the Factor XII of the coagulation system. As you know this protein is held inactive in circulation, but activates when it is outside the blood vessels. The second protein in the blood is Prekallikrein which is inactive. Then there is the High Molecular Weight Kininogen (HMWK). All 3 of these proteins will be carried out of the blood and into the tissues. This will cause the activation of several cascades across the coagulation, complement and contact activation system. This all starts with Factor XII becoming activated by Collagen outside the blood vessels. The Active Factor XII (XIIa) will kick off the coagulation cascade through the intrinsic pathway. This plays a role in clotting, but also in trapping pathogens in the coagulation of these factors. The second role of XIIa will be to convert Prekallikrein into Kallikrein. The Kallikrein will have 3 roles of its own. It will activate more Factor XII in a feedback loop. Then it will convert all the HMWK that entered the tissue into Bradykinin. It also will convert all the Low Molecular Weight Kininogen (LMWK) that is in the tissue into Kallidin which acts the same as Bradykinin. Both these molecules with Bradykinin and Kallidin will act on the Bradykinin receptors. The final role of the Kallikrein will be to activate C3 complement proteins into C3a. This helps stimulate the complement system to promote more inflammation and bind to pathogens.

The Bradykinin has a few roles of its own. It will bind to the Bradykinin receptors on Endothelial cells and promote more production of nitric oxide and PGI2. This will enhance the Vasodilation and Vascular Permeability. This causes more release of exudate into the tissue. Bradykinin will also bind to nerve receptors in the tissue and stimulate pain. Bradykinin has other effects, but we focused on those related to the topics we covered.

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