Hematology 425 Normal Coagulation & Hemostasis Russ Morrison December 4, 2006 11/27/2018

Coagulation & Hemostasis Hemostasis is defined as the combination of cellular and biochemical events that function to keep blood liquid within the veins and arteries, prevent blood loss from injuries by the formation of thrombi and reestablish blood flow during the healing process When this process is out of balance, thrombosis (clotting) or hemorrhage will threaten life 11/27/2018

Coagulation & Hemostasis It is the role of physicians and the laboratory to investigate the major hemostasis systems including Blood vessels Blood cells Plasma proteins The role of the investigation is to prevent, predict, diagnose and manage hemostatic disease 11/27/2018

Overview of Hemostasis For an overview of hemostasis, several factors must be discussed including Vascular intima Blood platelets Erythrocytes Neutrophils Monocytes Plasma coagulation system Fibrinolysis Each will be discussed as a separate component, but none act independently of the others or from the group 11/27/2018

Primary & Secondary Hemostasis Desquamation and small injuries to blood vessels Involves vascular intima and platelets Rapid, short-lived response Secondary Large injuries to blood vessels and tissues Involves platelets and the plasma coagulation system Dealyed, long term response 11/27/2018

Primary & Secondary Hemostasis Primary hemostasis mechanisms are triggered by small injuries to blood vessels (pinpricks) Or commonplace desquamation of dying or damaged endothelial cells In primary hemostasis, the blood vessel contracts to seal the wound and platelets fill the open space to form a plug While seeming to be trivial, defects in this process can cause fatal hemorrhagic disorders 11/27/2018

Primary & Secondary Hemostasis Secondary hemostasis is triggered by primary hemostasis mechanisms and is necessary to control bleeding from large wounds incurred through trauma, surgery or dental procedures The plasma coagulation system accomplishes secondary hemostasis by producing a fibrin thrombus Vascular intima and platelets are usually associated with 1o hemostasis and coagulation and fibrinolysis with 2o hemostasis, BUT all systems work together during hemostatic events 11/27/2018

Vascular Intima - Structure The innermost lining of blood vessels is a contiguous layer of endothelial cells These endothelial cells for a smooth, unbroken surface that promotes fluid passage of blood and prevents turbulence that might activate platelets and plasma enzymes A collagen-rich basement membrane and its surrounding layer of connective tissues support the endothelial cells Fibroblasts occupy the connective tissue layer and produce collagen Smooth muscle cells, intermixed with fibroblasts in arteries and arterioles (not veins, venules or capillaries), contract during primary hemostasis 11/27/2018

Vascular Intima - Procoagulant Intimal cells and the environment they are in play a primary role in hemostasis Any harmful local stimulus (mechanical or chemical) induces vasoconstriction in arteries and arterioles. The smooth muscle cells contract, the vascular lumen narrows (or closes) and blood flow to the injured site is minimized 11/27/2018

Vascular Intima - Procoagulant Second, the basement membrane and subendothelial connective tissues of arteries and veins are rich in collagen, an elastic protein that binds and activates platelets. Third, endothelial cells secrete vWF, a glycoprotein that is necessary for platelets to adhere to exposed subendothelial collagen in arterioles 11/27/2018

Vascular Intima - Procoagulant Fourth, upon activation, endothelial cells secrete and coat themselves with P-selectin, an adhesion molecule that promotes platelet and leukocyte binding. Additional adhesion molecules (ICAMs and PECAMs) that promote leukocyte binding are also secreted by endothelial cells Finally, subendothelial cells (smooth muscle cells and fibroblasts) support a constitutive surface protein called tissue factor 11/27/2018

Vascular Intima Exposed tissue factor activates the plasma coagulation system through factor VII Tissue factor also appears on the surface of endothelial cells and on blood-borne monocytes during inflammation While damaged vessels have PRO-COAGULANT properties, intact vessels prevent intravascular thrombosis and have ANTI-COAGULANT properties that act by several mechanisms 11/27/2018

Vascular Intima - Anticoagulant Endothelial cells are rhomboid and contiguous which provides a smooth inner surface that evens blood flow and prevents turbulance Endothelial cells also synthesize prostacyclin, a platelet activation inhibitor derived from arachidonic acid-prostaglandin pathway Prostacyclin prevents unnecessary or undesirable platelet activation in undamaged vessels 11/27/2018

Vascular Intima - Anticoagulant Nitric oxide is synthesized in endothelial cells, vascular smooth muscle cells, neutrophils and macrophages Nitric oxide regulates vasoconstriction and is essential in maintaining healthy arterioles Heparan sulfate is a glycosaminoglycan that slows plasma coagulation by activating antithrombin, a coagulation regulatory protein 11/27/2018

Vascular Intima - Anticoagulant Heparin is a pharmaceutical manufactured from pig gut tissues, that resembles heparan sulfate Heparin is used as a therapeutic to prevent propogation of thrombi that cause coronary thrombosis, strokes, DVTs and pulmonary thrombotic emboli Another important endothelial cell anticoagulant is tissue factor pathway inhibitor (TFPI) TFPI inactivates coagulation factor VIIa in the presence of factor Xa and controls the tissue factor (extrinsic) coagulation pathway 11/27/2018

Vascular Intima – Anticoagulant Finally, endothelial surface membranes contain thrombomodulin, a protein that activates the protein C pathway The protein C pathway then regulates the plasma coagulation mechanism by digesting activated factors V and VIII The coagulation pathways (intrinsic and extrinsic) will be discussed in detail later in this discussion 11/27/2018

Vascular Intima - Fibrinolytic ECs support fibrinolysis with two secretions, TPA and PAI-1 Tissue plasminogen activator (TPA) binds to polymerized fibrin and triggers activation of bound plasminogen to plasmin which ultimately digests the thrombus and restores blood flow Plasminogen activator inhibitor (PAI-1) inactivates TPA (and vice/versa) 11/27/2018

Vascular Intima The significance of the vascular intima to coagulation and hemostasis is recognized, but difficult to assess or to measure functional integrity Research is currently being directed toward evaluation of blood vessel disorders Hopefully, in the near future, we will gain protocols to assess the integrity of ECs, smooth muscle cells, fibroblasts and their collagen matrix 11/27/2018

Procoagulant Intima Structure Procoagulant Property Smooth muscle cells in arterioles and arteries Induce vasoconstriction Exposed basement membrane Collagen binds to vWF and platelets Damaged or activated endothelial cells Secrete vWF Exposed smooth muscle cells and fibroblasts Secrete adhesion molecules: P-selectin, ICAMS, PECAMS Endothelial cells in inflammation Tissue factor is induced by inflammatory process 11/27/2018

Anticoagulant Intima Anticoagulant properties of intact endothelial cells Rhomboid, presenting a smooth, contiguous surface Secrete platelet inhibitor, prostacyclin Secrete vascular relaxing factor, nitric oxide Secrete anticoagulant glycosaminoglycan, heparan sulfate Secrete coagulation pathway regulator, TFPI Maintain the protein C coagulation regulatory system activator, thrombomodulin on their surfaces 11/27/2018

Platelets Platelets are complex, metabolically active cells that interact with their environment and initiate hemostasis Platelets adhere, aggregate and secrete (table 42-3) Adhesion is the property of binding to nonplatelet surfaces such as subendothelial collagen Aggregation is the property of platelets binding to each other Secretion of platelet granule contents occurs during both adhesion and aggregation 11/27/2018

Platelet Adhesion Damaged or dead endothelial cells release their hold on the basement membrane leaving an area of turbulence that traps platelets In arterioles (where blood flow is rapid), vWF bridges the physical distance between platelets and exposed subendothelial collagen creating bonds that seal the platelet to the vessel wall In venules and veins, platelets directly bind the matrix proteins and vWF is unnecessary for platelet adhesion 11/27/2018

Platelet Adhesion Platelets only adhere for a few hours During adhesion, pltlts may secrete growth factors that stimulate fibroblast mitosis and aid in repair of subendothelial tissue Neighboring endothelial cells produce daughter cells that fill in the space occupied by the platelet, eventually forcing the platelet out This released platelet lives and circulates to repair other sites Adhesion of platelets is essential to life, individuals without good adhesion (vW disease) demonstrate mild to severe uncontrolled bleeding 11/27/2018

Platelet Aggregation With moderate to severe injury, platelets bind to each other to form platelet plugs which block the injury site and prevent further blood loss (fig 42-4) When pltlts aggregate, they expend their stored energy sources, lose membrane integrity and form an unstructured mass called a syncytium Once aggregated, pltlts can not disaggregate 11/27/2018

Platelet Aggregation In addition to plugging the site of injury, platelet aggregates shed microplatelet membrane particles rich in phospholipids and a variety of coagulation proteins These particles provide a localized environment that supports plasma coagulation Platelet aggregation is necessary to prevent blood loss from major wounds 11/27/2018

Platelet Aggregation To aggregate, pltlts depend upon an ample supply of vWF and fibrinogen Fibrinogen is an abundant coagulation protein that bridges natural platelet-to-platelet distances and encourages the formation of the platelet plug Fibrinogen is also the main structural component of a fibrin clot Fibrinogen deficiency (as vWF deficiency) is associated with hemorrhagic disease because both platelet aggregation and fibrin clot formation are deficient 11/27/2018

Platelet Secretion When platelets aggregate, they secrete their granular contents Granules contain A number of clot-promoting molecules called procoagulants Vasoconstrictors that cause blood vessels to contract Agonists that recruit and activate neighboring platelets 11/27/2018

Platelet Secretion Secretion occurs at the same time as adhesion and aggregation The substances secreted by platelets are essential to the plasma coagulation process In storage pool diseases, platelet granules contain diminished quantities of these substances Patients with storage pool disease suffer from easy bruising and a tendency to bleed freely after trauma, during surgery and after dental procedures 11/27/2018

Platelet Secretion There are relatively few tests available to measure platelet function Count and platelet morphology provides minimal, quantitative information A bleeding time screens platelet function as can the Dade-Behring PFA-100 Platelet aggregometry tests are semi-quantitative and technically demanding 11/27/2018

Hemostasis – Other Blood Cells Erythrocytes, monocytes and lymphocytes also participate in hemostasis RBCs add bulk and structural integrity to the fibrin clot In inflammation, monocytes and lymphocytes provide surface-borne tissue factor that triggers coagulation Leukocytes also have a series of membrane integrins and selectins that bind adhesion molecules and help stimulate the production of inflammatory materials that stimulate the wound healing process 11/27/2018

Plasma Coagulation There are at least 16 glycoproteins that function together to form a fibrin clot Most are trypsin-like enzymes called serine proteases The plasma coagulation system is complex and translates a diminutive physical or chemical event into a profound life-saving event (clot formation) 11/27/2018

Plasma Coagulation If even one of the plasma protein coagulants is missing in an individual, he/she is doomed to lifelong hemorrhage, chronic inflammation and transfusion dependence The plasma coagulation system which will be discussed includes normal plasma coagulation, plasma coagulation control and fibrinolysis 11/27/2018

Plasma Coagulation - Nomenclature Plasma contains a minimum of 16 procoagulants or clotting factors Most are glycoproteins synthesized in the liver (a few are made by monocytes, ECs and megakaryocytes Eight of the clotting factors are enzymes that circulate in an inactive form and are called zymogens 6 others are cofactors that bind and stabilize their respective enzymes 11/27/2018

Plasma Coagulation - Nomenclature During the thrombosis process, the procoagulants become activated and produce a localized thrombus (clot) At least 6 plasma glycoproteins serve as anticoagulants to keep the coagulation process regulated In 1958, the International Committee for the Standardization of the Nomenclature of Blood Clotting Factors named the plasma procoagulants using roman numerals The clotting factors were numbered in order of discovery 11/27/2018

Plasma Coagulation - Nomenclature When a procoagulant becomes activated, a lower case “a” appears behind the roman numeral (activated factor VII becomes factor VIIa) Both zymogens and cofactors become activated in the coagulation process Table 42-5 lists the coagulation factors, their functions, molecular weight, half-life and normal plasma concentration Knowledge of the coagulation factors (name and/or number) is essential clinical information that helps interpret lab tests and to design effective replacement therapies with deficiency-related hemorrhagic diseases 11/27/2018

Function of Plasma Procoagulants Plasma procoagulants may be either serine proteases or cofactors Serine proteases are proteolytic enzymes of the trypsin family and include the procoagulants thrombin (IIa), factors VIIa, IXa, Xa, Xia, and XIIa and prekallikrein Each has a reactive seryl amino acid residue in its active site and acts upon its substrate by hydrolyzing peptide bonds and digesting the primary backbone to produce small polypeptide products 11/27/2018

Function of Plasma Procoagulants These serine proteases are synthesized as inactive zymogens consisting of a single peptide chain Activation occurs when the zymogen is cleaved at one or more specific sites by the action of another protease during the coagulation process Activation is a localized process limited to the site of injury Generalized plasma activation of zymogens is called DIC, a condition with high morbidity that is often fatal 11/27/2018

Plasma Procoagulant Serine Proteases Inactive Zymogen Active Protease Cofactor Substrate Prothrombin (II) VIIa Fibrinogen VII Tissue factor IX, X IX IXa VIIIa X Xa Va Prothrombin XI XIa XII XIIa HMWK Prekalikrein Kalikrein HMWK – High Molecular Weight Kininogen 11/27/2018

Function of Plasma Cofactors Coagulation cofactors are tissue factor, factor V, factor VIII, and High Molecular Weight Kininogen (HMWK) Each cofactor binds a particular serine protease When the cofactor binds, the serine protease gains stability and increased reactivity 11/27/2018

Plasma Procoagulant Cofactors Inactive Form Active Form Binds Tissue Factor Exposed tissue factor VIIa V Va Xa VIII VIIIa IXa HMWK Kinin XIIa, prekallekrein 11/27/2018

Function of Add’l Components Remaining components of the plasma coagulation pathway are factor XIII, fibrinogen, calcium, vWF and phospholipids Vactor XIIIa is a transglutaminase that catalyzes the transfer of amino acids among the gamma chains of fibrin polymers The reaction cross-links fibrin polymers to add physical strength to the fibrin clot Factor XIII reacts with other plasma and cellular structural proteins and is essential for wound healing and tissue integrity 11/27/2018

Function of Add’l Components Fibrinogen is the primary substrate of the coagulation pathway When hydrolyzed by thrombin, fibrinogen polymerizes to form the primary structural protein of the fibrin clot vWF is a large glycoprotein that participates in platelet adhesion and transports the procoagulant factor VIII vWF is synthesized in megakaryocytes and endothelial cells 11/27/2018

Function of Add’l Components Coagulation reactions occur on the surface of platelet or endothelial cell membrane phospholipids, not in the fluid phase Serine proteases bind to negatively charged phospholipid surfaces through positively charged calcium ions Therefore, calcium is involved in the majority of the coagulation pathway reactions 11/27/2018

Fibrinogen Structure & Fibrin Formation Fibrinogen is the primary substrate of thrombin It is a large glycoprotein synthesized in the liver NL plasma concentration of fibrinogen is 200 to 400 mg/dL, the highest of all the plasma procoagulants Platelet granules absorb, transport and release fibrinogen 11/27/2018

vWF/Factor VIII Complex vWF is a multimeric glycoprotein composed of several subunits of 240,000 Da each The subunits are produced by endothelial cells and megakaryocytes where they combine to form molecules of 800K to 20M Da vWF molecules are stored in platelet alpha granules and in endothelial cells, where their storage sites are called Weibel-Palade bodies vWF molecules are released from storage into the plasma, with NL concentration of 7-10 ug/ml 11/27/2018

vWF/Factor VIII Complex vWF molecules carry receptor sites for platelets and collagen The primary platelet receptor site binds a platelet surface integrin, glycoprotein 1b/IX and fills the space between the platelet and exposed subendothelial collagen during platelet adhesion A secondary site binds another platelet integrin, glycoprotein IIb/IIIa A third site binds plasma procoagulant cofactor, factor VIII 11/27/2018

vWF/Factor VIII Complex Factor VIII is produced by the liver and from other tissues Its source (as well as the source of factor IX) is the X chromosome Factor VIII has a molecular mass of 260K Da and circulates bound to vWF Free factor VIII is unstable and cannot be detected in plasma 11/27/2018

vWF/Factor VIII Complex Factor VIII is a labile factor that deteriorates over a period of hours in stored blood even when still bound to vWF Individuals with hemophilia A have diminished factor VIII activity, but normla vWF levels Because factor VIII depends on vWF for stability, people with vWF disease have both diminished vWF and diminished factor VIII activity, but factor VII levels do not drop to clinically significant levels 11/27/2018

Vitamin K-Dependent Prothrombin Group Prothrombin; factors VII, IX, and X and the regulatory proteins, protein C and protein S are all vitamin K dependent This group is called the prothrombin group because of their structural resemblance to prothrombin The net negative charge of these molecules enable them to bind ionic calcium The bound calcium enables the vitamin K-dependent proteins to bind to phospholipids Phospholipid binding is essential to coagulation reactions 11/27/2018

Vitamin K-Dependent Prothrombin Group When vitamin K is deficient or a vitamin K antagonist is present, this group of plasma coagulation factors is released from the liver without the carboxylation that produces the net negative charge Without the charge, they cannot bind to calcium and participate in the coatulation reaction Vitamin K antagonism is the basis of oral anticoagulant therapy (warfarins/ coumadin) 11/27/2018

The Tissue Factor Pathway Coagulation is triggered by the exposure of tissue factor to plasma proteins Activated factor VII (VIIa) binds tissue factor in the presence of phospholipid and Ca and triggers the activation of the zymogens, beginning with factors IX and X Activated factor IX (IXa) binds VIIIa on phospholipid surfaces and that complex activates factor X (fig 42-10) 11/27/2018

The Tissue Factor Pathway Factor Xa binds vactor Va on phopholipid surfaces (fig 42-11 and 42-12) This Xa-Va complex activates prothrombin in a multistep hydrolytic process that releases a peptide fragment from prothrombin called prothrombin 1+2 Activated prothrombin is called thrombin Thrombin cleaves fibrinopeptides A and B from plasma fibrinogen causing the formation of fibrin polymer Fibrin polymer is stabilized by the cross-linking action of factor XIIIa (fig 42-7) 11/27/2018

The Tissue Factor Pathway This tissue factor pathway is characterized by multimolecular complex formation The first complex is composed of tissue factor, VIIa, phospholipid and Ca2+ The second is IXa, VIIIa, phospholipid and Ca2+ (also called tenase) The third complex is Xa, Va, phospholipid and Ca2+ (also called prothrombinase) 11/27/2018

Coagulation Cascade Complexes Components Function First complex VIIa, tissue factor, phospho-lipid and Ca2+ Cleaves IX and X Second complex IXa, VIIIa, phospholipid,Ca2+ Cleaves X, called “tenase” Third complex Xa, Va, phospholipid and Ca2+ Cleaves pro-thrombin, called “prothrombinase” 11/27/2018

Factor XI Activation Pathway Thrombin also activates factor XI Activated XI (Xia) activates factor IX and the reaction proceeds as described in the prior slides This alternative pathway is essential to normal coagulation as seen in case of moderate to severe hemorrhage in folks with factor XI deficiency (Rosenthal syndrome) 11/27/2018

Contact Factors The “contact factor” complex composed of XIIa, HMWK and prekallikrein also activates factor XI Contact factors do not have an in vivo procoagulant function, but respond to the presence of negatively charged particulate surfaces such as nonsiliconized glass or the aPTT test reagent Foreign material, such as stents or valves may activate contact factors in vivo and cause thrombosis 11/27/2018

Extrinsic, Intrinsic and Common Pathways Before 1992, coagulation experts called the activation of factor XII the primary step in coagulation The reason was that factor XII could be found in blood and tissue factor could not be found in blood The reaction steps that begin with factor XII and culminate in fibrin polymerization was called the intrinsic pathway The coagulation factors of the intrinsic pathway, in order of reaction were XII, prekalikrein, HMWK, XI, IX, VIII, X, V, prothrombin and fibriogen 11/27/2018

Extrinsic, Intrinsic and Common Pathways The tissue factor pathway was called the extrinsic pathway The extrinsic pathway includes factors VII, X, V, prothrombin and fibrinogen Neither factor VIII nor factor IX was included in the extrinsic pathway because their contribution was bypassed in the prothrombin time test (PT) The PT was used to measure the integrity of the extrinsic pathway, while the aPTT was used to measure the intrinsic pathway 11/27/2018

Extrinsic, Intrinsic and Common Pathways These two pathways had factors X, V, prothrombin, and fibrinogen in common The steps involving these “common” factors was termed the common pathway Terms intrinsic and extrinsic pathways no longer fit with current understanding of coagulation, but one must be aware of the terms as they are still used in clinical discussion of coagulation processes See fig 42-10 11/27/2018

The coagulation cascade The coagulation cascade. Legend: HMWK = High molecular weight kininogen, PK = Prekallikrein, TFPI = Tissue factor pathway inhibitor. Black arrow = conversion/activation of factor. Red arrows = action of inhibitors. Blue arrows = reactions catalysed by activated factor. Grey arrow = various functions of thrombin. 11/27/2018

Coagulation Regulatory Mechanisms Fig. 42-13 illustrates the regulatory points of the coagulation mechamism We will summarize the points and then discuss selected ones in more detail 11/27/2018

Coagulation Regulatory Mechanisms Name Function MM (Da) Half-life (hours) Mean plasma concen-tration Tissue factor pathway in-hibitor With Xa,binds TF-VIIa 33K unknown 60-80 ng/mL Thrombo-modulin EC surface receptor for thrombin 450K Does not circulate None Protein C Serine Protease 62K 7-9 2-6 ug/mL 11/27/2018

Coagulation Regulatory Mechanisms Name Function MM (Da) Half-life (hours) Mean plasma concen-tration Protein S cofactor 75K unknown 20-25 ug/mL Antithrom-bin SERPIN 58K 68 24-40 mg/dL Heparin cofactor II 65k 60 30-70 ug/mL α1-anti-trypsin 60K 250 mg/dL α2-Macro-globulin 725K 150-400 ,g/dL MM = molecular mass, SERPIN = serine protease inhibitor 11/27/2018

Coagulation Regulatory Mechanisms Tissue Factor Pathway Inhibitor Fig 42-13 & 14, TFPI inactivates factor VIIa It is the action of TFPI that makes the tissue factor-factor VIIa reaction one with a short half-life Coagulation pathway amplification would occur primarily through factor XI 11/27/2018

Coagulation Regulatory Mechanisms Protein C Regulatory System, fig 42-15 During thrombosis, thrombin cleaves fibrinogen and activates factors V, VIII, XI and XIII In intact normal vessels, where coagulation is unwanted, thrombin binds the EC membrane protein thrombomodulin and triggers the Protein C system, an anticoagulant regulatory system 11/27/2018

Coagulation Regulatory Mechanisms The complex of thrombin-thrombomodulin activates the plasma zymogen, protein C Activated protein C (APC) then binds free plasma protein S The stabilized APC-protein S complex hydrolyzes and inactivates factors Va and VIIIa Protein S is synthesized in the liver and circulates in the plasma in two forms 11/27/2018

Coagulation Regulatory Mechanisms The two forms of circulating Protein S are: Free Covalently bound to the complement control protein, C4b-binding protein Bound protein S cannot participate in the protein C anticoagulant pathway, only free plasma protein S In inflammatory conditions the complexed Protein S increases which decreases free Protein S Deficiencies of Proteins C & S may be associated with recurrent venous thromboembolic disease 11/27/2018

Coagulation Regulatory Mechanisms Serine Protease Inhibitors (SERPINs), fig 42-16 Antithrombin is a SERPIN that binds and neutralizes thrombin and factors IXa, Xa, XIa, and XIIa Anththrombin was the first of the plasma coagulation regulatory proteins to be identified and the first to be assayed routinely in the clinical laboratory Other SERPINs include heparin cofactor II, α1-ahtitrypsin and α2-macroglobulin 11/27/2018

Coagulation Regulatory Mechanisms Antithrombin and heparin cofactor II both require heparin for effective anticoagulant activity Unfractionated therapeutic heparin is a heterogenous glycosaminoglycan Therapeutic unfractionated heparin increases antithrombin’s ability to neutralize thrombin and other serine proteases by 1000 times and simultaneously binds thrombin and antithrombin 11/27/2018

Fibrinolysis The last stage of coagulation is fibrinolysis Fibrinolysis begins a few hours after fibrin polymerization and cross-linking The precise mechanism of fibrinolysis is the subject of current research, but in general, consists of systematic hydrolytic digestion of cross-linked fibrin olymers by bound plasmin (fig 42-17) Bound plasminogen is activated by TPA several hours after thrombus formation and restores normal blood flow through repaired vessels 11/27/2018

Fibrinolysis - Plasminogen Plasminogen is a 90K Da zymogen produced in the liver Plasminogen becomes bound to fibrin at the time of fibrin polymerization Bound plasminogen becomes converted to an active plasmin molecule through proteolysis The active form, plasmin, is a serine protease that systematically digests the fibrin polymer 11/27/2018

Fibrinolysis - Plasminogen Plasmin is also capable of digesting fibrinogen and factors V and VIII, but is localized to and bound to fibrin which prevents that activity ECs secrete tissue plasminogen activator (TPA), which hycrolizes bound plasminogen and initiates fibrinolysis TPA, forms covalent bonds with fibrin during polymerization and segregates at the surface of the thrombus with plasminogen where it begins the digestion process over time 11/27/2018

Fibrinolysis - Plasminogen Free TPA circulates bound to nhibitors such as PAI-1 and is cleared from plasma Synthetic recombinant TPA is a therapeutic drug widely used to dissolve pathologic clots that form in venous and arterial thrombotic disease 11/27/2018

Fibrinolysis - Urokinase Urinary tract epithelial cells secrete an “intrinsic” plasminogen activator called urokinase Small amounts of urokinase circulate in the plasma and become incorporated into the mix of fibrin-bound plasminogen and TPA at the time of thrombus formation Urokinase does not bind firmly to fibrin and has a minor physiologic effect Like TPA, urokinase is widely used to dissolve clots in heart attacks, strokes and DVTs 11/27/2018

Fibrinolysis – Plasminogen Activator Inhibitor-1 TPA and urokinase are prevented from activating free, fluid-phase plasminogen by an inhibitor, PAI-1 PAI-1 is secreted from endothelium with TPA Only at times of endothelial cell activation, such as following trauma, does the level of TPA secretion exceed that of PAI-1 to initiate fibrinolysis 11/27/2018

Fibrinolysis – α2-antiplasmin Bound plasmin digests clots and restores blood vessel patency Free plasmin digests plasma fibrinogen, factor V, factor VIII, and fibronectin, causing potentially fatal primary fibrinolysis α2-antiplasmin is a free plasma protein that rapidly and irreversibly binds free plasmin α2-antiplasmin also becomes cross-linked to fibrin during polymerization rendering fibrin resistant to digestion by plasmin 11/27/2018

Fibrin Degradation Products and D-dimer Fibrinolysis proceeds in a methodical fashion, producing a series of identifiable fibrin fragments (X,Y,D,E, D-D) Several of these fragments inhibit hemostasis by preventing platelet activation and by hindering fibrin polymerization These fragments can be detected by laboratory assay 11/27/2018

Fibrin Degradation Products and D-dimer The D-D fragment is called D-dimer and is separately detectable by immunoassay to provide evidence of cross-linking Pathologic degradation of fibrinogen, as occurs when free plasmin is present, yields D and E fragments but no detectable D-dimer 11/27/2018