Hematology 425 Normal Coagulation & Hemostasis

1. Hematology 425 Normal Coagulation & Hemostasis Russ Morrison December 4, 2006

2. 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

3. 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

4. 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

5. Primary 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 Primary & Secondary Hemostasis

6. 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

7. 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

8. 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

9. 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

10. 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. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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)

18. 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

19. Procoagulant Intima

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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)

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. Plasma Procoagulant Serine Proteases

39. 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

40. Plasma Procoagulant Cofactors

41. 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

42. 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

43. 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

44. 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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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)