Introduction to hemostasis 1 Flashcards
- Explain what is meant by the term hemostasis, and list the different components involved in the hemostatic process.
Hemostasis is the mechanism used by the body to stop bleeding from a damaged blood vessel.
It occurs via complex interactions involving the blood vessel wall, the platelets, and the coagulation system, leading to formation of a blood clot. The system needs to respond rapidly in response to injury to prevent life‐ threatening hemorrhage. At the same time, the process must be tightly regulated to limit clot formation to the site of injury and to avoid pathologic clotting (thrombosis), which can lead to loss of blood flow (ischemia) to tissues. Hemostasis is a complicated process. To better understand it, we will break it down into its component parts. While we may think of these components as acting separately and sequentially, it is important to remember that this is an artificial distinction and that in reality everything is occurring simultaneously, with crosstalk among the different parts.
Explain Platelet pulg formation (primary hemostasis):
At the time of injury what released from endothelial cells leads to vasoconstriction?
Primary hemostasis
Secondary hemostasis
- Identify which coagulation factors are serine proteases and which are cofactors.
What factor is th exception? What is its function?
Almost all the enzymes in the coagulation cascade are serine proteases, related to trypsin and/or chymotrypsin, which function by cleaving their targets at arginyl residues**.
The inactive precursor proteins that are activated through cleavage into active enzymes are called zymogens. Zymogens that become active serine proteases in the cascade include factor XII, Prekallikrein, factor XI, factor IX, factor X, factor VII, and factor II (prothrombin).
- Identify which coagulation factors are serine proteases and which are cofactors.
Do they have intrinsic enzymatic activity?
In addition to the enzymes in the cascade, there are several cofactors that are essential for initiating or accelerating enzymatic reactions but lack intrinsic enzyme activity themselves. The cofactors are thought to work by bringing the components together and orienting them properly to make the reaction more efficient.
Cofactors in the coagulation cascade include tissue factor, factor VIII, factor V, and high molecular weight kininogen (HMWK).
Tissue factor (TF) is not ordinarily expressed on cells in direct contact with the blood (endothelial cells, leukocytes). It is, however, expressed on the fibroblasts and smooth muscle cells surrounding blood vessels. With blood vessel injury, factor VIIa from the plasma will become exposed to tissue factor, leading to activation of the extrinsic pathway. Tissue factor can also become expressed on endothelial cells and monocytic blood cells under conditions of stress or injury, or when stimulated by lipopolysaccharide (LPS) or other proinflammatory agents. Factors VIII and V are both non‐ enzymatic procofactors with significant sequence homology to each other. Both are cleaved by thrombin to their active form, allowing them to participate in the tenase and prothrombinase complexes.
HMWK participates as a cofactor with the contact factors, factor XIIa, kallikrein, and factor XI.
- Describe how fibrinogen is converted to fibrin by thrombin, leading to formation of an insoluble fibrin network, and how factor XIII functions in stabilizing the forming clot.
Factor XIII covalently cross-link _____ domains to stabilize and strenghten the developing clot.
Fibrinogen is the soluble plasma protein that is cleaved by thrombin into its insoluble form, fibrin, which then participates in forming the actual blood clot.
- Fibrinogen is made up of 3 pairs of polypeptide chains (2 Aα‐chains, 2 Bβ‐chains, and 2 γ‐chains) arranged into identical half molecules in an elongated polypeptide composed of three globules: a central globule (labelled the E domain) containing the N‐terminal domain of all the polypeptides, and globules on each end of the molecule, designated D domains.
- With activation, thrombin cleaves off two small peptides, fibrinopeptide A and fibrinopeptide B.
The release of fibrinopeptide A leads to exposure of a site on the E domain that aligns non‐covalently with a complementary site in the D domain of another fibrin molecule to form overlapping fibrils.
The subsequent cleavage and release of fibrinopeptide B allows increasing aggregation of the growing fibrils.
Factor XIIIa then covalently cross‐links adjacent D domains, stabilizing and strengthening the developing clot.
- Describe how fibrinogen is converted to fibrin by thrombin, leading to formation of an insoluble fibrin network, and how factor XIII functions in stabilizing the forming clot.
Factor XIII cross-linkage
- List the two main functions of von Willebrand factor (VWF) in coagulation
Serves as the carrier protein for _____ in the plasma.
Why do patients with low levels of VWF have a low circulating factor VIII?
Finally, von Willebrand factor (VWF), while playing a critical role in platelet adhesion and aggregation as we will see later, also plays an important role in the coagulation cascade serving as the carrier protein for factor VIII in the plasma.
- VWF is a large, multimeric protein that is produced and stored in Weibel‐Palade bodies in endothelial cells and in α‐granules of platelets.
- In the circulating plasma, it binds to and protects factor VIII, significantly prolonging its half‐life (12 hrs vs 2 hrs). Patients with severe deficiency of VWF have low levels of circulating factor VIII, leading to a bleeding disorder similar to classical hemophilia A (factor VIII deficiency). They have a coexistent platelet function defect, leading to a very severe bleeding problem.
- Explain the role of vitamin K in coagulation, and list the factors that are vitamin K dependent.
What do the Gla residues bind, and what is the effect of this?
What does a vitamin K deficiency leads to?
A subset of this group of proteases are the vitamin‐K dependent factors, including factors II, VII, IX, and X, along with the anticoagulant protein C. Protein S is also vitamin K dependent, though it functions in coagulation as a cofactor for protein C and not as a serine protease.
-These proteins share significant homology and are likely derived evolutionarily from a common ancestor. A special property that they all share is that they contain a “Gla” domain. This domain contains several (9 to 13) glutamic acid residues that undergo post‐translational modification to γ‐carboxy glutamic acid (“Gla” residues).
These Gla residues bind calcium, leading to shape change
of the protein that allows binding to an anionic phospholipid surface which is necessary for normal protein function. This γ‐carboxylation is carried out by the enzyme γ‐glutamyl carboxylase in the liver.
Vitamin K is required for generation of the precursor for the reaction, so vitamin K deficiency leads to inability to make the Gla residues, resulting in non‐functional protein. As a result, vitamin K deficiency is an important cause of bleeding to consider, as we will see later. Also, you will learn about an important anticoagulation drug (Warfarin, brand name Coumadin) used to treat people at risk for thrombosis that targets this pathway, as illustrated in the diagram.
- Explain the role of vitamin K in coagulation, and list the factors that are vitamin K dependent.
Why does hemorrhagic disease of the newborn happens?
- Later found to be due to Vitamin K deficiency
- Reasons newborns are prone to develop:
- Decreased stores
- Low levels in breast milk
- Developing gut flora
- List and explain the role of the different components of the intrinsic tenase, extrinsic tenase, and prothrombinase complexes.
Enzyme complexes: protease + cofactor + phospholipid surface + calcium
A common theme you may note as we look at the coagulation cascade is the situation where an activated enzyme combines with a cofactor on a negatively charged phospholipid surface in the presence of calcium, forming an enzyme complex that accelerates the speed of a reaction several hundred‐ to several thousand‐fold. These complexes are often referred to as “_____ase.” Examples include:
- Tissue factor (a cofactor) combines with factor VIIa (a serine protease) on a phospholipid surface in the presence of calcium to bind to and activate either factor IX or factor X. This is called “extrinsic tenase” (or Xase), since it is part of the extrinsic pathway.
Alternatively, two members of the intrinsic pathway, Factor IXa (a serine protease) and factor VIIIa (a cofactor), combine with phospholipid and calcium to bind and activate factor X. This complex is called “intrinsic tenase.”
Activated factor X (Xa), a serine protease, can then combine with factor Va (a cofactor), on a phospholipid surface in the presence of calcium, to bind to and activate prothrombin (factor II) to thrombin (factor IIa). This is known as the “prothrombinase complex.”
- List the components of the extrinsic and intrinsic coagulation pathways and relate these pathways to the PT and APTT coagulation screening tests
Because the prothrombin time was not informative for patients with hemophilia, modified versions of the test were developed in the 1950s using “partial thromboplastins” that distinguished normal and hemophilic plasma. We now know that these alternate methods lacked tissue factor, which was the critical component contained in thromboplastin that accelerated clotting. This eventually lead to the development of the “partial thromboplastin time”, or PTT, the other major coagulation screening test in use today, which measures activity of what we now know as the intrinsic pathway. It is called the “intrinsic” pathway because all the factors required to have a normal result are intrinsic to the plasma itself, as opposed to the “extrinsic” pathway, which requires the presence of tissue thromboplastin. To optimize the test, the reaction is “activated” by addition of an anionic substrate such as kaolin, silica, ellagic acid, or celite.
Hence the term “activated partial thromboplastin time”, or aPTT, sometimes written APTT. The intrinsic pathway is also sometimes known as the “contact pathway,” since it is initiated by contact with an anionic surface, or the “accessory pathway,” since in current models it is felt to have more of a supportive/amplifying role to coagulation that is initiated by the extrinsic pathway.
- List the components of the extrinsic and intrinsic coagulation pathways and relate these pathways to the PT and APTT coagulation screening tests
- Explain the current concept of the process of coagulation, describing what occurs during the initiation, amplification, and propagation phases.
With this model, coagulation is initiated with vascular disruption that leads to exposure of plasma to tissue factor (TF).
In the plasma, a small amount (~1 %) of circulating factor VII can normally be found in its activated (VIIa) form. Free factor VIIa that is not bound to TF is inactive, which is why the small amount of circulating VIIa doesn’t initiate clotting.
With exposure of tissue factor (TF) on the cell surface, it can bind factor VIIa in the presence of calcium, and the TF‐VIIa (extrinsic tenase) complex can then bind factor X, leading to production of minute amounts of factor Xa.
TF‐VIIa also converts factor IX to factor IXa. The TF‐VIIa complex can also generate more VIIa to amplify the process. This process, termed the initiation phase of coagulation, produces sufficient quantities of Xa to start the process of clot generation. Factor V can be slowly activated by factor Xa. Factor Xa then binds to factor Va and forms the prothrombinase complex, which generates small amounts of thrombin.
Initiation
Initiation
Initiation
Amplification
- The amplification phase takes place on the surface of platelets which have adhered to the exposed subendothelium and been activated.
- In this phase, the initial procoagulant signal is amplified by small amounts of thrombin generated on TF‐bearing cells.
- Thrombin activates Factors V and VIII to Va and VIIIa, respectively. Upon platelet activation, factors Va and VIIIa bind to the platelet surface. Xa can then form the prothrombinase complex with Va to generate small amounts of thrombin from prothrombin.
Amplification
Propagation
The propagation phase of coagulation begins with assembly of procoagulant complexes on the cell surface. The intrinsic tenase complex (factor VIIIa and factor IXa) activates more factor X on the platelet surface. The rate of activation of factor X by the intrinsic tenase complex is about 50‐100 times faster than the extrinsic tenase complex, making formation of the intrinsic tenase complex critical for producing a sufficient quantity of Xa to initiate coagulation. Factor Xa then rapidly binds to factor Va (generated by thrombin in the amplification phase), forming the prothrombinase complex, which initiates conversion of prothrombin to thrombin, producing the thrombin burst necessary for fibrin‐ clot formation. Thrombin cleaves fibrinogen to form fibrin, leading to formation of the fibrin clot, and activates factor XIII to XIIIa, which covalently cross‐links and stabilizes the forming clot.
–>A fibrin latticework develops between and around the activated platelets which have adhered and aggregated at the site of injury. The activated platelets provide a surface for the coagulation reactions to continue to occur, generating even more thrombin. Platelets also secrete the contents of their granules, which contain many of the components of coagulation to provide more substrate for the reactions to take place. Simultaneously, anticoagulation and fibrinolytic reactions are occurring, which will rapidly quench this process and prevent its spread beyond the site of injury, as we will discuss in part 2.
Coagulation
- Explain why thrombin is considered the central enzyme in blood coagulation.
At this point, it is important to emphasize the central role that thrombin plays in blood coagulation.
Not only does it cleave fibrinogen to fibrin to form the actual clot.
- It also cleaves and activates the precofactors V and VIII to Va and VIIIa, which can then participate in the prothrombinase and intrinsic tenase complexes, respectively.
- It also activates factor XI to XIa, which can then go on to generate more factor IXa to participate in the intrinsic tenase complex.
- This all results in amplification of the coagulation response and production of a burst of thrombin sufficient to form the clot.
- Thrombin also cleaves and activates factor XIII, which covalently links the fibrin to form a more stable clot.
- In addition to its role in the coagulation cascade, thrombin is the most potent known activator of platelets.
You will learn more about the essential role of platelets in clot formation in a subsequent lecture, but suffice it to say that one of the results of platelet activation is the translocation of the anionic phospholipid phosphatidylserine from the inner leaflet of the platelet cell membrane to the outer leaflet. Exposure provides a surface for the coagulation reactions to take place, greatly increasing their efficiency.
