What is FMD?

Brachial artery scanning to measure FMD.

Flow-Mediated Dilatation is the increase in calibre of a blood vessel in response to increased blood flow. This was first described by Schretzenmayr. FMD is endothelium dependent, wherein the endothelium acts as a mechanotransducer that senses changes in shear stress and then determines the release of dilatators. Several dilator factors have been proposed to be involved in FMD: prostaglandins , ATP or an endothelium-derived hyperpolarizing factor, and nitric oxide (NO). An increased release of NO in response to increases in shear stress causes dilatation of underlying smooth muscle of conduit arteries.

Why is FMD significant?

A decreased FMD reflects endothelial dysfunction and may predict coronary artery disease in susceptible patients. The brachial artery FMD can be measured by ultrasound scan and acts as surrogate for the coronary vasculature which can only be assessed by more invasive methods.

How is FMD related to platelet reactivity?

Another effect of NO released by the endothelium under increased shear stress is decrease of platelet reactivity. Does it follow, therefore, that if decreased FMD determines endothelial dysfunction and decreased NO release, it also determines an increased platelet reactivity?

A study by Shechter, et.al. explores the association between platelet reactivity and brachial artery FMD in controls (without established cardiovascular disease) and in patients with acute myocardial infarction. Platelet reactivity was measured by conventional aggregometry and by the IMPACT-R machine. The study concludes that FMD is inversely correlated to platelet reactivity in both controls and AMI patients. An abstract of the said study can be found here.

Dohle body in the cytoplasm of a granulocyte.

The Inherited Macro-thrombocytopenias are autosomal dominant disorders characterized by mild to moderate thrombocytopenia with large platelets and varying degrees of platelet dysfunction, usually an absence or reduction of the secondary wave of platelet aggregation resulting in little or no bleeding. Thrombopenic symptoms are purpura, ecchymoses, epistaxis and heavy menstrual bleeding. Some may also have leukocyte inclusions, interstitial nephritis, sensorineural hearing loss and cataracts.  Mutations in the heavy chain IIA MYH9, a prominent non-muscle myosin, result in a spectrum of disorders with variable manifestations.

May-Heggelin anomaly (MHA) is characterized by macrothrombocytopenia and presence of Döhle bodies in the cytoplasm of leukocytes. Most of the time, MHA requires no treatment but in extreme cases, a platelet transfusion may be beneficial.

Fechtner syndrome includes a macrothrombocytopenia with smaller calibre platelets than MHA, leukocyte inclusions, interstitial glomerulonephritis, sensorineural deafness, and cataracts. Treatment includes specific management of the components (e.g. lens replacement for cataract) and platelet transfusions if contemplating surgery.

Sebastian syndrome is purely hematologic in manifestation. It involves macrothrombocytopenia which is usually asymptomatic and leukocyte inclusion bodies that are smaller than those seen in MHA, may be numerous and are generally difficult to stain.

Epstein syndrome has nephritis, hearing loss, platelet adhesion and aggregation defects but no leukocyte inclusions are demonstrated. Eckstein syndrome has nephritis and deafness but no platelet dysfunction. Enyeart syndrome has thrombocytopenia and giant platelets with inclusions but no leukocyte inclusions.

Cytoplasmic inclusions are formed when the unstable abnormally dimerized MYHIIA protein precipitates with the normal MYHIIA.

Giant platelet.

Abnormal dimerization also brings about failure to properly organize the cytoskeleton of megakaryocytes, which causes giant platelets and thrombocytopenia.

Since these disorders are autosomal dominant with phenotypic consequences that are not readily apparent, screening tests for siblings or offspring of known affected individuals may be an important step. In this regard, Impact-R can be of much use. It is a cone and platelet analyzer that qualitatively determines platelet adhesion and aggregation. A peripheral smear alone may not predict the need for platelet transfusions in patients about to undergo surgery. Impact-R could also be of much use in further research for these conditions especially in answering the question, “How is adhesion and aggregation affected by abnormal platelet size?”

Inherited platelet disorders are rare conditions that are not usually encountered in clinical practice. However, the study of the pathophysiology has led to a better understanding of platelet biochemistry and physiology. They may be subdivided into the following:

1. Platelet Membrane Disorders
2. Platelet Granule Disorders
3. Macrothrombocytopenias
4. Platelet Signaling Disorders

The Platelet Membrane Disorders:

Glanzmann’s Thrombasthenia is a rare disorder where platelets can carry out biochemical reactions but are unable to form aggregates. This is an autosomal recessive trait where platelets have absent or dysfunctional GpIIb/IIIa complexes.

Smooth inactivated platelet (blue) with spiky activated platelets (light blue). ©2000 Dennis Kunkel, Ph.D.

In a normal platelet, there are about 50,000 of these complexes in the membrane. When platelets are activated, the complex binds fibrinogen, which in turn bind to GpIIb/IIIa complexes on other platelets, resulting in multicellular aggregates. Platelets are normal in size, shape and number. Patients with Glanzmann’s Thrombasthenia have mucosal bleeding throughout life and may even have severe bleeding episodes requiring platelet transfusions.

Bernard-Soulier Syndrome is another rare autosomal recessive disorder caused by mutations of the GpIb/IX/V complex. This complex is the main receptor for von Willebrand’s factor, which anchors platelets to exposed subendothelium in cases of endothelial injury and under high shear stress. The platelets are abnormally large and the count is low. Patients present with muco-cutaneous bleeding and prolonged bleeding time. They may require platelet transfusions and sometimes respond favourably to Desmopressin.

Pseudo- or Platelet type von Willebrand Disease is an autosomal dominant disorder arising from mutations of the GpIba polypeptide that make the platelet hypersensitive to vWF. Mucosal bleeding and borderline thrombocytopenia are noted. Pseudo-vWD resembles Type IIB vWD. An accurate diagnosis is necessary since treatment differs, Pseudo-vWD requiring platelet transfusions while Type IIB vWD requires vWF transfusion.

ADP receptor Deficiency is an autosomal recessive disorder involving the ADP receptor P₂Y₁₂. ADP released from damaged tissues and activated platelets plays a role in platelet aggregation through the mediation of receptors P₂Y₁ and P₂Y₁₂.   P₂Y₁ initiates platelet response to ADP while P₂Y₁₂ forms and sustains large aggregates. Patients, therefore, have mild bleeding but are susceptible to posttraumatic and post-surgical blood loss.

Collagen receptor deficiency involves two receptors, an integrin protein, GpIa/IIa, and a non-integrin protein, GpVI. This deficiency is still under intense study.

Platelet function studies that show how platelets behave in vitro can be very helpful in diagnosis of these conditions. One of these is studying the platelet aggregates formed when blood is exposed to polystyrene in flow conditions. The IMPACT-R machine is one laboratory equipment that uses the cone and plate principal.

A novel method to test for platelet function may be an important adjunct in the diagnosis of and research about ITP. This is the CPA or Cone and Plate Analyzer (IMPACT-R) which tests for platelet adhesion and aggregation under simulated arterial flow conditions.

Immune Thrombocytopenic Purpura (ITP) otherwise known as Idiopathic Thrombocytopenic Purpura, is an acquired platelet disorder. This involves immune-mediated destruction of platelets as well as inhibition of platelet release from the megakaryocyte. The exact nature of the immune dysfunction is not known but is usually associated with infections and auto-immune disorders. Common infections causing ITP are Human Immunodeficiency Virus and Hepatitis C.  Systemic Lupus Erythematosus is the most common auto-immune disorder implicated.

ITP usually presents as an acute disease in children and runs a more chronic course in adults. Mucocutaneous bleeding in the gastrointestinal tract (gum bleeding, oral mucosal bleeding, etc.), and in the female reproductive tract (heavy menstrual bleeding) are seen. Patients also note ecchymoses and petechiae. Thrombocytopenia on a routine CBC may be the only sign

Blood blister under the tongue

prompting a consultation. Life-threatening bleeding very rarely occurs and is usually heralded by oral blood blisters and retinal hemorrhage.

A careful and comprehensive history and physical examination will reveal a high index of suspicion for ITP. Laboratory testing to confirm the diagnosis includes: complete blood count with a peripheral blood smear, bone marrow biopsy, clotting time and bleeding time. The peripheral smear will show large platelets with normal morphology. Laboratory tests to look for the secondary causes of ITP are recommended. These are: testing for HIV and Hepatitis C, serologic testing for SLE.  If anemia is present, a Coomb’s test is done to rule out Evan’s Syndrome. This syndrome is a combination of ITP and autoimmune hemolytic anemia.

The new Impact-R cone and plate analyzer can be of use in screening for ITP and also for monitoring therapeutic response.

Treatment for ITP depends on severity. Mild ITP, or those without significant bleeding symptoms and severe thrombocytopenia, can be treated in an outpatient setting. Agents include Prednisone, Rh₀(D) immune globulin, intravenous gammaglobulin (IVIgG). Severe ITP requires hospitalization and treatment with high-dose glucocorticoids, anti-Rh₀(D), IVIgG, and immunosuppressive agents such Rituximab. Patients who relapse after medical therapy may benefit from a splenectomy.

Blood cells are produced in the bone marrow. The three main components of blood are the red blood cells which carry oxygen, white blood cells or leukocytes which fight infection and platelets also called thrombocytes which assist in the formation of blood clot.

When a blood vessel is damaged platelets adhere to the surface of damaged vessel wall and release chemicals. The chemicals attract more platelets as well as red blood cells in order to form a clot or thrombus. As the clot grows the blood vessel narrows, thereby decreasing the blood loss. This process is called coagulation.

Normal platelet counts are in the range of 150, 000 to 350, 000 platelets per micro liter. Thrombocytopenia or low blood platelets are the disorders in which there are not enough platelets in the blood. When the platelet count is decreased the body is unable to form blood clots and is therefore unable to control the bleeding. Bruising and bleeding can occur from relatively little trauma. When the platelets count gets below 10, 000 platelets per micro liter, bleeding can develop even without significant trauma.

Chemotherapy induced thrombocytopenia is a disorder that develops as an adverse effect of chemotherapy. Cancer drugs not only kill cancer cells, they can also damage the platelet forming cells in the bone marrow. The severity of this disorder depends on the type of chemotherapy and the duration of treatment. Fortunately chemotherapy induced thrombocytopenia or low blood platelets can be managed with platelet transfusions, additional medications such as blood cell growth factors or with blood stem cell transplants.

Other common causes of thrombocytopenia or low blood platelets are ITP (immune thrombocytopenic purpura) and heparin induced thrombocytopenia. In immune thrombocytopenic purpura, anti platelet antibodies coat the platelets and destroy them, while heparin induced thrombocytopenia is caused by the formation of abnormal antibodies that activates platelets.

Impact-R has evolved as a blessing in disguise for the thrombocytopenia hit populations as it provides a very effective screening test for the timely detection of the patients suffering from thrombocytopenia. It has markedly decreased the morbidity and mortality associated with this disorder.

Before discussing the causes of low blood platelets or thrombocytopenia, I feel necessary to mention the grave consequences associated with low blood platelets. The dominant features of thrombocytopenia include petechial cutaneous bleeding, intracranial bleeding and oozing from mucosal surfaces.

Causes can be divided into three main groups;

• Decreased production
• Increased destruction
• Un replaced loss or dilution of platelets

The characteristic findings of thrombocytopenia are decreased platelet count and prolonged bleeding time. Bone marrow aspiration is helpful in the cases of thrombocytopenia. It reveals decreased megakaryocytes (immature form of platelets) when caused by decreased platelet production and increased megakaryocytes when caused by increased platelet destruction.

Now I would like to discuss few common causes of low blood platelets;

• Irradiation, exposure to drugs or chemical causes decreased production of the platelets.
• Acute leukemia in which there is decreased platelet production due to replacement of bone marrow by blast cells.
• Myelophthisis results in low blood platelets because the bone marrow is replaced by tumor cells.
• Aplastic anemia can also be the cause of low blood platelets. It may be caused by exposure to toxic agents like benzene or due to autoimmune destruction by cytotoxic T cells.
• Circulating blood platelets are lost or destroyed when they are sequestered by spleen, thus decreasing the blood platelet count.
• Dilution of platelets occurs in multiple transfusions, where there is relatively deficiency of platelets and thus low blood platelets.
• Disseminated intravascular coagulation, commonly known as consumptive coagulopathy results in consumption of almost all the platelets in the blood.
• Low blood platelets can be secondary to other disease such as acquired immunodeficiency syndrome or systemic lupus erythematosus.
• Immune thrombocytopenic purpura is a very common autoimmune disorder of the platelets occurring due to formation of anti platelet antibodies in the blood, which damage the platelets and decreases their count.

All the above mentioned causes of thrombocytopenia can be diagnosed well before time by the use of Impact-R platelet analyzer. This kit uses the very basic principles of platelet aggregation and adhesion and can be used to screen the cases of thrombocytopenia due to any cause.