6 - Recombinant Coagulation Factors & Thrombolytic Agents

Question 1

What is the difference between thrombosis and hemostatis?

Answer 1

• Thrombosis = formation and propagation of a blood clot w/in vasculature
• Hemostasis = stoppage of blood flow

Question 2

Describe thrombosis under normal conditions

Answer 2

Thrombus is confined to the immediate area of injury and doesn’t obstruct flow to critical areas (unless the blood vessel lumen is already diminished)

Question 3

Describe thrombosis under pathologic conditions

Answer 3

• A thrombus can propagate into normal vessels to obstruct flow in the vessels
• It can obliterate valves and other structures that are essential to normal hemodynamic function

Question 4

Describe the process of thrombosis

Answer 4

• Vascular injury => immediate local cellular response takes place => attracts platelets to migrate to the area of injury
• • Platelets secrete several cellular factors and mediators to promote thrombus formation
• During thrombus formation, circulating prothrombin is activated to the active clotting factor and thrombin, fibrinogen is activated to fibrin by the newly activated thrombin and fibrin is then formed into the fibrin matrix
• 3 main components involved in blood clot = platelets, thrombin, fibrin
• • Each of these components can be a therapeutic target

Question 5

What is thrombosis involved in?

Answer 5

1. Blood flow and the blood vessel
2. Platelet-vessel interactions related to disruption of endothelium
3. Coagulation system – cellular elements (platelets), protein elements (coagulation factors and vWF)

Question 6

What is thrombolysis?

Answer 6

• Opposite process of thrombosis
• Involves fibrin-specific activators to activate plasminogen at the fibrin surface
• Thrombolytic agents available today are serine proteases that work by converting plasminogen to the natural fibrinolytic agent plasmin
• Plasmin lyses clots by breaking down the fibrinogen and fibrin in a clot

Question 7

Define tPA and describe its function

Answer 7

• Tissue plasminogen activator (tPA) = naturally occurring fibrinolytic agent found in vascular endothelial cells and is involved in the balance between thrombolysis and thrombosis
• • Exhibits significant fibrin specificity and affinity
• At the site of the thrombus, the binding of tPA and plasminogen to the fibrin surface induces a conformational change that facilitates the conversion of plasminogen to plasmin and dissolves the clot
• • Fibrin-specific agents produce limited plasminogen conversion in the absence of fibrin (ex: alteplase (tPA), reteplase, tenecteplase)
• • Non-fibrin-specific agents catalyze systemic fibrinolysis (streptokinase)

Question 8

Clinical application of recombinant coagulation factors

Answer 8

Hemophilia

Question 9

Clinical applications of recombinant thrombolytic agents

Answer 9

• Acute ischemic stroke
• Acute MI
• Acute PE
• Restoration of central venous access devices

Question 10

Describe primary coagulation

Answer 10

• Platelets from plasma bind to exposed collagen via surface glycoprotein IIb/IIIa receptor w/ vWF
• Platelets release their granules (ADP and TxA2) to activate more platelets
• Fibrinogen crosslinks adjacent platelets forming a platelet plug at the site of injury

Question 11

Describe secondary coagulation

Answer 11

Proteins in plasma (coagulation factors) respond in a complex cascade which eventually leads to fibrin formation (strengthens the platelet plug)

Question 12

What does the coagulation cascade consist of?

Answer 12

2 pathways:

1. Intrinsic pathway (contact activation pathway)
2. Extrinsic pathway (tissue factor pathway)

Question 13

Hemophilia – definition, types, diagnosis

Answer 13

• Genetic disease; rare; more common in males
• Involves long bleeding times; some are internal causing damage to organs/ tissues (especially ankles, knees and elbows)
• Little clotting factor which is needed for normal blood clotting
• Hemophilia A (classical hemophilia) -> little to no clotting factor VIII, 90% of people w/ the disorder have this type
• Hemophilia B (Christmas disease) -> missing or having low levels of clotting factor IX
• If hemophilia suspected:
• • Personal and family medical hx
• • Physical exam
• • Blood tests – PT (extrinsic pathway) and APTT (intrinsic and common pathway)

Question 14

Hemophilia cause

Answer 14

• Defect in one of the genes that determines how the body makes blood clotting factors VIII or IX -> located on X chromosomes
• Males – abnormal gene on X chromosome results in hemophilia
• Females must have abnormal gene on both X chromosomes (very rare)
• • Females can be a “carrier” if they have an abnormal gene on one of their X chromosomes -> can pass the gene to offspring

Question 15

Hemophilia severity index

Answer 15

• Can range from mild to severe, depending on how much clotting factor is in the blood
• Mild = 5-30% of normal factor
• Moderate = 1-5% of normal factor
• Severe = < 1% of normal factor

Question 16

Hemophilia sx

Answer 16

• Easy bruising
• Excessive bleeding
• • External (obvious) – bleeding in the mouth from a cut or bite, unexplained nosebleeds, heavy bleeding from a minor cut
• • Internal – blood in urine or stool, bleeding in joints or brain

Question 17

What is factor replacement therapy? Where do they come from?

Answer 17

• Basic tx to stop or prevent bleeding in px w/ hemophilia A or B
• Involves infusion of factor VIII and IX concentrates to prevent or control bleeding
• Concentrates come from 2 sources – human plasma (plasma-derived) and genetically engineered cell line made by DNA technology (recombinant)

Question 18

Factor VIII

Answer 18

• Plasma protein
• Synthesized as a single-chain polypeptide of 2332 amino acids
• First gen factor VIII concentrates have bovine serum albumin that is used as a stabilizer; second gen = human albumin-free; third gen = no human or animal protein used in culture medium or manufacturing process

Question 19

What are the considerations for changing the dose of recombinant factor VIII? What is the usual dose?

Answer 19

• Depends on:
• • Severity of deficiency
• • Severity of hemorrhage
• • Presence of inhibitors
• • Desired increase in factor III activity
• Dosage required (IU) = body weight (kg) * desired increase in factor VIII (%) * 0.5 IU/kg%

Question 20

What are the considerations for changing the dose of recombinant factor IX? What is the usual dose?

Answer 20

• Dosage and duration of tx w/ BeneFix depends on:
• • Severity of factor IX deficiency
• • Location and extent of bleeding
• • Clinical condition
• • Pt age
• • Desired recovery in factor IX
• Factor IX dosage required (IU) = body weight (kg) * desired increase in factor IX (%) * 1.2 IU/kg%

Question 21

Recombinant factor VIIa

Answer 21

• Developed b/c some px w/ hemophilia A or B will develop Ab (inhibitors) to factor VIII or factor IX respectively after using recombinant factor VIII or factor IX
• Expressed in a single-chain form and is spontaneously activated to factor VIIa during purification
• Very similar to plasma-derived factor VIIa w/ regard to amino acid sequence, carbohydrate composition and gamma-carboxylation

Question 22

Describe the structure of tPA

Answer 22

• 527 amino acids
• 17 disulfide bridges
• 4 N-linked glycosylation sites at residues 117, 184, 218, and 448

Question 23

Structure-function relationships of tPA

Answer 23

• F domain -> residues 4-50 homologous w/ the finger domain mediating the fibrin affinity of fibronectin
• E domain -> residues 50-87 homologous w/ epidermal growth factor
• K1 domain residues 87-176 and K2 domain residues 176-256 share a high degree of homology w/ the 5 kringles of plasminogen
• P domain -> residues 276-527 are a serine protease region w/ active-site residues His322, Asp371, and Ser478
• Binding to fibrin = F domain and K2 domain
• Rapid clearance in vivo w/ initial t1/2 of 6 minutes in human = E domain and carb side chains
• Enzymatic activity = P domain
• Rapid inhibition by plasminogen activator inhibitor-1 (PAI-1) = Lys296-His-Arg-Arg299

Question 24

Alteplase – what does it do? what is it used for? what are the 2 possible treatment regimens?

Answer 24

• Contains 4 distinct regions that contribute to its structure-function activity (fibrinogen finger, EGF growth factor domain, Kringle domains and proteinase domain)
• Binds to fibrin in a thrombus (clot) and converts plasminogen to plasmin, thereby initiating local fibrinolysis
• Used for:
• • Lyses of occlusive coronary artery thrombi
• • Reduction in mortality
• • Improve cardiac function
• • Reduce incidence of congestive heart failure
• 2 treatment regimens – 90-minute accelerated infusion (recommended up to 6 h of AMI sx) or 3 h infusion (effective up to 12 h of AMI sx)

Question 25

Function of Cathflo

Answer 25

Used to restore the function of central venous access devices that have been occluded by thrombus formation

Question 26

Tenecteplase -- structure, uses, why is it called TNK?

Answer 26

- 527 amino acid glycoprotein - Modified form of the endogenous tissue plasminogen activator (tPA) molecule - Includes 4 domains (fibronectin finger, EGF growth factor domain, Kringle domains, and proteinase domain) - Used in acute myocardial infarction; 40 mg as single bolus over 5-10 second period - Called TNK b/c t = threonine, N = asparagine, K = lysine

Question 27

Describe the modifications from anteplase to make tenecteplase. Advantages and what does each change cause?

Answer 27

* *Switch Thr with Asn at 103; switch Asn with Gly at 117, switch “Ala-Ala-Ala-Ala” for “Lys-His-Arg-Arg” at 296-299 - - Greater fibrin specificity than alteplase - - Longer plasma t1/2 than alteplase - - Resistance to PAI-1 (plasminogen activator inhibitor) - Substitution at 103 w/ asparagine means that glycosylation occurs at this position - Substitution at 117 w/ glutamine means that the high mannose sugar residue is absent at 117 position - - Both mutations occur w/in the kringle 1 domain (increase plasma t1/2 vs. tPA) - Tetra-alanine substitution at amino acids 296-299 in the protease domain - - Increased fibrin specificity and greater resistance to plasminogen activator inhibitor-1 (PAI-1)

Question 28

Reasons to develop 2nd generation of recombinant thrombolytic agents

Answer 28

- Rapid clearance of recombinant tPA (alteplase) from the liver - First gen of recombinant tPA may cause some general fibrinolysis although it is administered locally - Has relatively complicated administration procedure

Question 29

Reteplase -- structure, use, advantages over alteplase and tenecteplase

Answer 29

- 335 amino acid deletion variant of natural tPA, which consists of 527 amino acids - Contains 2 domains (protease and Kringle 2) - - Kringle 2 domain & lack of finger domain => low fibrin binding affinity - - Protease domain => fibrin specificity - Non-glycosylated protein - Used in STEMI as slow IV infusion; second dose given 30 mins after first dose - Advantages over alteplase and tenecteplase = less complicated procedure than alteplase and has an indication > 12 h after sx (alteplase and TNK do not)

Question 30

General monitoring of thrombolytic agents

Answer 30

- aPTT (activated partial thromboplastin time) - Complete blood cells count - Signs and sx of bleeding; bleeding associated w/ thrombolytic agents can be divided into 2 broad categories: 1. Internal (intracranial, retroperitoneal sites, GI, genitourinary) 2. Superficial (observed mainly at invaded or disturbed sites; also including epistaxis/ nose bleeding & hematuria/ blood cell in urine) - ECG