Blood Coagulation & Platelet Activation PDF

Blood Coagulation & Platelet Activation Fadi Khasawneh, Ph.D. (361) 221-0755 [email protected] 1 Learning Outcomes At the end of these lectures, you should be able to: Explain hemostasis, the...

Blood Coagulation & Platelet Activation Fadi Khasawneh, Ph.D. (361) 221-0755 [email protected] 1 Learning Outcomes At the end of these lectures, you should be able to: Explain hemostasis, the physiology of blood coagulation and physiological prevention of coagulation Understand and explain the various platelet signaling/activation pathways (e.g., GPCRs-mediated), their role in clot formation and how they are initiated. Explain the pathophysiological relevance of coagulation abnormalities, and thrombosis development. List the body’s endogenous inhibitors of coagulation. Identify the theoretical strategies for producing antiplatelet agents 2 1  Introduction -The physiological systems that control blood fluidity are both complex and elegant. - Blood must remain fluid within the vasculature and yet clot quickly when exposed to non-endothelial surfaces at sites of vascular injury. Thus, humans have developed a well-regulated system of hemostasis to keep the blood fluid and clot-free in normal vessels and to form a localized plug rapidly in injured vessels. - When intravascular blood clot/thrombi (normal hemostatic processes are activated inappropriately) do occur, a system of fibrinolysis is activated to restore fluidity. In the normal situation, a delicate balance prevents both thrombosis and hemorrhage and allows physiological fibrinolysis without excess pathological fibrinogenolysis. 3 Blood Blood volume ~ 5-6 L Plasma/ Serum volume ~ 3-4 L Blood cells – RBC – Platelets – WBC Proteins – Albumin – Globulin – Clotting factor proteins Lipids Functions – Transport of O2, nutrients, waste – Defense 4 2  Blood, Plasma, and Serum Blood – Plasma + blood cells Plasma – Serum + fibrinogen + clotting factors Serum – Water + serum proteins + antibodies etc 5 How to prepare plasma/serum from blood? Plasma – Add an anticoagulant like sodium citrate or heparin and centrifuge the blood  supernatant will be plasma Serum – Keep the whole blood after collection without adding any anticoagulant until the blood clots, and centrifuge  supernatant will be serum 6 3  Blood Vessels Artery – Arterioles Vein – Venules 7 Blood Cells - Erythrocytes (RBC): 4.2-6.1 million/μl platelets - Leucocytes (WBC): 5-10 k/μl - Platelets (thrombocytes): 250-500 k/μl 8 4  Hemostasis Spontaneous arrest of blood loss from injured blood vessels and is essential to life. Keep the blood fluid and clot free. Thrombosis a pathological process in which thrombi occlude blood flow to vital organs Coagulation Conversion of fluid blood into a gel  clot Activation  clot Inappropriate activation  thrombus formation Decreased activation (Deactivation)  bleeding Anticoagulants Substances which prevent coagulation 9 Anticoagulants in the Nature Anopheles: thrombin-directed anticoagulants (in the saliva) Culex : Factor Xa-directed anticoagulants (in the saliva) Animals depending on a diet of fresh blood have evolved mechanisms that interfere with the coagulation process of the blood donor. 10 5  Anticoagulants in the Nature Leech: Hirudo medicinalis. These animals make: 1. Hirudin: thrombin inhibitor = Lepirudin: recombinant hirudin 2. Antistasin: Factor Xa inhibitor Animals depending on a diet of fresh blood have evolved mechanisms that interfere with the coagulation process of the blood donor. 11 “Dracula’s” Anticoagulant 12 6  Why Study Coagulation & Anticoagulants? Coagulation – Protective in function – Also causes clinical problems Hemophilia Preventing Coagulation – Treat conditions mediated by abnormal coagulation Disseminated intravascular coagulation disorder ? ? 13 Physiology of Coagulation Initiated by injury or stasis Platelets & Coagulation factors play major role Coagulation factors (I-XIII) Activated platelet, Fibrin, RBC  thrombus/clot formation 14 7  Coagulation Factors and Pathways Coagulation factors are plasma glycoproteins produced by hepatocytes. Most are serine proteases in an inactive zymogen form that is converted to the active form by proteolytic cleavage of a specific peptide bond. In the coagulation cascade, an active coagulation factor cleaves and activates its substrate. The goal of the coagulation cascades is to convert soluble fibrinogen to insoluble fibrin. Extrinsic pathway - activated by tissue factor expression. Intrinsic pathway - activated by contact of Factor XII with an anionic surface (collagen). 15 Coagulation Factors and Pathways (2) Platelet procoagulant activity is required for efficient blood clotting. Platelets provide a phospholipid surface for the assembly of Ca2+- dependent coagulation complexes. Thrombin in turn activates platelets to aggregate. Platelet activation and blood coagulation are inter-dependent events for clot formation. 16 8  Coagulation Pathways Contact Activation pathway or Intrinsic pathway (stimulus from factors in the blood) Tissue Factor pathway or Extrinsic pathway (stimulus from factors outside the blood) Contribute to the formation of a clot 17 Clot Formation Links for videos that describe the process of hemostasis and clot formation http://www.mhhe.com/biosci/esp/2002_general/Esp/folder_structure/tr/m1/s7/trm1s7 _3.htm http://www.hopkinsmedicine.org/hematology/Coagulation.swf 18 9  Steps Involved in Clot Formation Vasoconstriction (triggered by endothelin secretion at site of injury from endothelial cells). Platelet adhesion Platelet activation Platelet aggregation  plug formation Fibrin Formation (mesh) RBC and additional platelet incorporation  clot formation 19 Platelets - Platelets are cell fragments with a life span of around 10 days, that arise by budding from megakaryocytes in the bone marrow. Platelets are small, membrane-bound discs contain cytoplasm but lack nuclei (anucleated). Platelets play important role in hemostasis and thrombosis. - The platelet surface contains many glycoproteins (GP) that serve as adhesive receptors for adhesive proteins present in blood and in the subendothelial matrices of the blood vessel wall (e.g., fibrinogen, fibronectin, von Willebrand Factor (vWF)). 20 10  Platelets (2) In addition, platelets also contain receptors for interaction with physiological stimuli such as: 1. ADP, 2. thrombin, 3. thromboxane A2 (TXA2), 4. collagen and 5. epinephrine. “Activated” platelets undergo a series of reactions that lead to the formation of hemostatic plugs and thrombi. 21 Platelet Adhesion, Activation & Aggregation (1) Platelets normally do not adhere to shielded/masked subendothelium of a blood vessel. However, when a blood vessel is damaged, platelets adhere to the exposed subendothelium, which initiates their activation processes. This adhesion is mediated by the platelet membrane glycoproteins (GP) known as GPVI and GPIb. GPVI binds to collagen and von Willebrand factor (vWF) binds to and GPIb. Vascular injury causes endothelial denudation (loss), thus, collagen is exposed. 22 11  Platelet Secretion (Release Reaction) Activated platelet release contents from their dense granules which include potent platelet agonists/activators such as: ADP and serotonin; TXA2 - Activated platelets will also make TXA2. 23 Platelet Adhesion, Activation & Aggregation Activation of other platelets results from binding of ADP and TXA2 to their respective receptors (i.e., G-protein coupled receptors; abbreviated [GPCRs]). P2Y1 and P2Y12 are receptors (GPCRs) for ADP (adenosine diphosphate); Thromboxane A2 (TxA2) is the major product of COX-1 involved in platelet activation through binding to the thromboxane A2 receptor (GPCR); when stimulated by agonists, these receptors activate the fibrinogen-binding glycoprotein GPIIb/IIIa to promote platelet aggregation (platelet-to-platelet interaction). 24 12  Platelet Adhesion, Activation & Aggregation Activation of other platelets results from binding of ADP and TXA2 to their respective receptors (i.e., G-protein coupled receptors; abbreviated [GPCRs]). PAR1 and PAR4 are protease-activated receptors (GPCRs) that are activated by thrombin (clotting factor IIa); when stimulated by agonists, these receptors activate the fibrinogen-binding glycoprotein GPIIb/IIIa to promote platelet aggregation (platelet-to-platelet interaction). 25 Platelet Adhesion, Activation & Aggregation (2) The symmetrical fibrinogen molecule will act as a molecular bridge linking the activated platelets together (e.g., crosslink two platelets). 26 13  Platelet Adhesion, Activation & Aggregation (3) Prostaglandin I2 (PGI2), synthesized by endothelial cells, inhibits platelet activation through binding to its receptor (GPCR) and raising cAMP levels. Thus, cAMP itself is a platelet inhibitor (platelets cannot aggregate and form a clot even in the presence of an agonist or stimuli). Phosphodiesterases are enzymes that will degrade or breakdown cAMP, and reverse it’s inhibitory effects on platelet activation 27 Thromboxane A2 Formation by COX Cycloxygenase (COX) 28 14  Platelet Activation by Thromboxane A2 TXA2 activates platelets by increasing intracellular calcium levels 29 Platelet Activation* (B) Resting platelet Activated spread platelet Activated contracted platelet Scanning electron micrographs of resting platelets (B), a platelet undergoing cell spreading shortly after cell activation (C), and a fully activated platelet after actin filament bundling and crosslinking and myosin contraction (lamelopodia projections) (D). *FYI 30 15  Platelet Activation by ADP (Adenosine DiPhosphate) and Thrombin (e.g., P2Y12) ADP activates platelets by lowering the levels of the platelet inhibitor cAMP and by increasing intracellular calcium levels. 31 Clot Formation √ 32 16  Any questions? The End 33 17