5 - Coagulation Factors Flashcards
Define Thrombosis
may be defined as the formation and propagation of a blood clot within the vasculature
What is hemostasis?
refers to the stoppage of blood flow
What is coagulation?
it’s a complex process where the blood forms solid clots in response to blood vessel damage
What is thrombosis an important part of?
the normal hemostatic response that limits hemorrhage caused by vascular injury
Under normal conditions, a thrombus is confined to the immediate areas of injury and dose not obstruct flow to critical areas, unless blood vessel lumen is already diminished, as it is in __________
atherosclerosis
What can a clot cause ?
- Acute myocardial infarction (AMI)
- Deep vein thrombosis (DVT)
- Pulmonary embolism (PE)
- Acute ischemic stroke (AIS)
- Acute peripheral arterial occlusion
- Occlusion of indwelling catheters
Thrombosis is a complex process and involved in which 3 factors?
- Blood flow and the blood vessel
- Platelet-vessel interactions related to the disruption of the endothelium
- Coagulation system
- cellular elements (platelets)
- protein elements (coagulation factors and mediators to promote thrombus formation)
When a vascular injures, an immediate local cellular response takes place which attracts ______ to migrate to the area of injury.
platelets
What do platelets do?
Platelets secrete several cellular factors and mediators to promote thrombus formation
During thrombus formation, circulating prothrombin is activated to the acting clotting factor _______.
thrombin
_______ is activated to fibrin by the newly activated thrombin.
Fibrinogen
What does fibrin do?
It is then formed into the fibrin matrix.
What are the 3 main components involved in blood clotting?
- platelets
- thrombin
- fibrin
Each of the 3 components (platelets, thrombin, and fibrin) can be therapeutic targets:
List 3 drugs that can inhibit platelet activation and aggregation.
- aspirin
- glycoprotein (GP) 2b/3a inhibitors
- clopidogrel
______ gathers in the fibrin matrix.
Plasminogen
What does fibrin-bound plasminogen do?
it will be converted by thrombolytic drugs to plasmin to initiate thrombolysis
Thrombolysis is the ______ process of thrombosis
opposite
What does Thrombolysis involve?
It involves fibrin-specific activators to a activate plasminogen at the fibrin surface.
What are the thrombolytic agents available today?
They are serine proteases that work by converting plasminogen to the natural fibrinolytic agent plasmin.
How does plasmin lyse clots?
by breaking down the fibrinogen and fibrin in a clot
List 1 naturally occurring fibrinolytic agent.
Tissue plasminogen activator (tPA)
What is tPA involved in?
(it is a naturally occurring fibrinolytic agent) found in vascular endothelial cells and is involved in the balance between thrombosis and thrombolysis
What does thrombosis exhibit?
Significant fibrin specificity and affinity
How does the binding of tPA dissolve a clot?
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 ______ of fibrin
presence
List some examples of fibrin-specific agents
- Alteplase (tPA)
- Reteplase
- Tenecteplase
What do non-fibrin-specific agents do?
They catalyze systemic fibrinolysis
List an example of a non-fibrin-specific agent
Streptokinase
What is hemophilia?
a rare bleeding disorder in which the blood doesn’t clot normally.
What is the problem in Hemophilia A?
genetic mutation in factor 8 gene
What is the problem in Hemophilia B?
lack of factor 9
What is the clinical application of recombinant coagulation factors?
Hemophilia (A or B)
What are some clinical applications of recombinant thrombolytic agents?
- Acute ischemic stroke
- Acute myocardial infarction (STEMI)
- Acute pulmonary embolism
- Restoration of central venous access devices (ex. dialysis)
Describe primary coagulation
Platelets from plasma bind to the exposed collagen via their surface Glycoprotein 2b/3a receptor with von Willebrand factor (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.
Describe secondary coagulation
proteins in plasma (coagulation factors) respond in a complex cascade which eventually leads to fibrin formation (strengthens the platelet plug).
The coagulation cascade consists of which two pathways?
- The intrinsic pathway (contact activation pathway)
- The extrinsic pathway (tissue factor pathway)
Describe Hemophilia
- Rare condition
- Inherited
- Usually occurs 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 is ______ hemophilia
classical
Describe Hemophilia A
- little to no clotting factor 8
- 90% of people with the disorder have this type
Hemophilia B is called _______ disease
Christmas
Describe Hemophilia B
- missing or have low levels of clotting factor 9
- 10% of people with the disorder have this type
- named after Steven Christmas, a Canadian who in 1952 was the first person to be diagnosed with this distinct form of hemophilia
What is done if hemophilia is suspected?
- Personal and family medical history
- Physical exam
- Blood tests
- Prothrombin time (PT) - extrinsic pathway
- Activated partial thromboplastin time (APTT) - intrinsic and common pathway
What causes hemophilia in general?
Defect in one of the genes that determines how the body makes blood clotting factors 8 or 9 –> located on X chromosomes
What causes hemophilia in males?
abnormal gene on X chromosome results in hemophilia
What causes hemophilia in 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 onto her children
Describe the severity index of hemophilia
- can range from mild to severe
- depends on how much clotting factor is in the blood
How much clotting factor is in MILD hemophilia ?
5-30% of normal factor
How much clotting factor is in MODERATE hemophilia ?
1-5% of normal factor
How much clotting factor is in SEVERE hemophilia ?
less than 1% of normal factor
What are the symptoms of Hemophilia ?
- Easy bruising
- Excessive bleeding (external or internal)
What are external bleeding symptoms?
Obvious:
- bleeding in the mouth from a cut or bite
- unexplained nosebleeds
- heavy bleeding from a minor cut
What are internal bleeding symptoms?
Hidden within the body:
- blood in urine or stool
- bleeding in joints or brain
What is factor replacement therapy?
- It is the basic treatment to stop or prevent bleeding in patients with hemophilia A and B.
- It involves the infusion of Factor 8 and 9 concentrates to prevent or control bleeding.
Concentrates of factors 8 and 9 come from which 2 sources?
- Human plasma (plasma-derived)
- Genetically engineered cell line made by DNA technology (recombinant)
Describe Factor 8
- plasma protein
- synthesized as a single chain polypeptide for 2332 amino acids
- circulates as an heterodimer of the 80 kDa light chain and a variable (90-210 kDa) heavy chain in a metal ion-dependent complex
What is the risk of Recombinate (human albumin as stabilizer) ?
risk of human disease
What is the risk of Kogenate or Helixate or Refacto ?
no risk of passing human disease but not very stable
uses sucrose as stabilizer
What is the advantage of Advate ?
More stable !
uses Trehalose as stabilizer
Dosage of recombinant Factor 9 must be individualized to the needs of the patient. It depends on which conditions:
- The severity of the deficiency
- The severity of the hemorrhage
- The presence of inhibitors
- The desired increase in Factor 3 activity
There are two factor 9’s: What are they produced in?
- CHO (only recombinant)
- Plasma (purified from the blood)
What amino acids are at 148 in CHO Factor 9?
Ala
What amino acids are at 148 in Plasma Factor 9?
Thr/Ala
Describe the carboxylation of CHO factor 9
40% is under carboxylated, lacking gamma-carboxylation at Glu40
Describe the carboxylation of Plasma factor 9
12 gamma-carboxyglutamic acid
What does the dose of recombinant Factor 9 depend on?
- the severity of factor 9 deficiency
- the location and extent of bleeding
- the clinical condition
- patient age
- the desired recovery of factor 9
Why was recombinant factor 7a developed ?
Because:
- 15-20% of patients with Hemophilia A
- 2-5% of patients with Hemophilia B
will develop antibodies (inhibitors) to factor 8 or 9 respectively after using recombinant factor 8 or 9.
Where is recombinant factor 7a produced in?
Baby hamster kidney cells
Recombinant factor 7a is expressed in a _____ chain form and is spontaneously activated to factor 7a during purification.
single
Recombinant factor 7a is very similar to plasma-derived factor 7a with regard to ?
amino acid sequence, carbohydrate composition and gamma-carboxylation
What is tPA?
Tissue Plasminogen Activator
-A protein involved in the breakdown of blood clots. It is a serine protease found on endothelial cells.
As an enzyme, what does tPA catalyze ?
The conversion of plasminogen to plasmin
What does the structure of tPA include ?
- consists of 527 amino acids
- with 17 disulfide bridges
- there are 4 N-linked glycosylation sites at residues 117, 184, 218 and 448
tPA:
Describe the F domain
residues 4 to 50, homologous with the finger domain mediating the fibrin affinity of fibronectin
tPA:
Describe the E domain
residues 50 to 87, homologous with epidermal growth factor
tPA:
Describe K1 domain
residues 87 to 176
tPA:
Describe K2 domain
residues 176 to 256
tPA:
What do K1 and K2 domain share?
a high degree of homology with the 5 cringes of plasminogen
tPA:
Describe the P domain
residues 276 to 527, a serine protease region with the active site residues His322, Asp371 and ser478
tPA:
What domains are responsible for binding to fibrin?
F domain and K2 domain
tPA:
What is involved in rapid clearance in vivo with an initial half life of 6 minutes in human?
E domain and carbohydrate side chains
tPA:
What domain is involved in enzymatic activity ?
P domain
tPA:
What is involved in rapid inhibition by plasminogen activator inhibitor-1 ?
Lys296-His-Arg-Arg299
**if we modify this sequence and can make it resistant to plasminogen activator inhibitor 1 or to increase half life
tPA:
What happens if we remove F and E domain?
more resistant to clearance in blood, increased half life
What are types of Alteplase?
rtPA, Activase, Cathflow
What is Alteplase produced in?
Chinese hamster ovary cells (CHO cells)
Alteplase is an enzyme that consists of ___ amino acids
527
Alteplase contains 4 distinct regions that contribute to it’s structure-function activity. What are they?
- Fibronectin Finger
- EGF growth factor domain
- Kringle domains
- Proteinase domain
What does alteplase do?
binds to fibrin in a thrombus (clot) and converts plasminogen to plasmin, thereby initiating local fibrinolysis
In an Acute MI:
What is alteplase used for?
- Lyses of occlusive coronary artery thrombi
- Reduction in mortality
- Improve cardiac function
- Reduce incidence of congestive heart failure
Alteplase has to be used within ___ hr of MI
12
Alteplase has 2 treatment regimens: a 90 minute accelerated infusion and a 3 hour infusion
Describe the 90 minute accelerated infusion
**don’t need to know doses
-recommend up to 6 hours of AMI symptoms
Alteplase has 2 treatment regimens: a 90 minute accelerated infusion and a 3 hour infusion
Describe the 3 hour infusion
(still 100mg total)
Effective up to 12 hours of AMI symptoms
What is Cathflo ?
Used to restore the function of central venous access devices that have been occluded by thrombus formation
What is TNKase?
Tenecteplase is a 527 amino acid glycoprotein
TNKase is a modified form of ?
the endogenous tissue plasminogen activation (tPA) molecule
What are the 4 domains of TNKase ?
- fibronectin finger
- EGF growth factor domain
- kringle domains
- proteinase domain
TNKase is produced in ____ cells
CHO
Describe the TNKase use in an AMI (acute myocardial infarction)
- 40mg as a single bolus over a 5-10 second period
- Never more than 50 mg
KNOW slide 37
okay
For TNKase, replace the K with __ ______ so can be resistant to plasminogen activation inhibitor 1
4 alanine
TNKase:
What substitution causes glycosylation occurs at 103 position?
a sub of threonine 103 with asparagine means that glycosylation occurs at 103 position
TNKase:
What sub means that the high mannose sugar residue is absent at 117 position?
sub of asparagus 117 with glutamine
TNKase:
What do mutations within the kringle 1 domain do?
- increase plasma half life (T50)
- 5 to 7 times longer half life vs tPA allowing for singles 5s bolus administration
TNKase:
What does a tetra-alanine substitution at amino acids 296-299 in the protease domain cause ?
- Increased fibrin specificity (14 fold greater fibrin selectivity over tPA)
- Greater resistance to plasminogen activator inhibitor-1 (PAL-1) - 80 fold more resistant to PAL-1 than tPA
What are the reasons to develop the second generation of recombinant thrombolytic agents?
- Rapid clearance of recombinant tPA (Alteplase) from the liver
- The first generation of recombinant tPA may cause some general fibrinolysis although it is administered locally
- It has relatively complicated administration procedure
What is Reteplase?
It is a 355 amino acid deletion variant of natural t-PA, which consists of 527 amino acids
What 2 domains does Reteplase have?
- Protease
- Kringle 2
Reteplase:
What leads to low fibrin binding affinity?
The kringle 2 domain and the lack of the finger domain lead to low fibrin binding affinity
Reteplase:
What leads to fibrin specificity?
the protease domain
Reteplase:
What type of protein is it?
non-glycoslylated protein
Reteplase:
Is synthesized in ____ as a single chain about 40 kDa peptide
E. coli
Reteplase:
How is it used in acute myocardial infarction in adults (STEMI) ?
- Two doses of 10 unites (18.1 mg) as slow intravenous infusion not to exceed two minutes.
- The second dose is given 30 minutes after the first dose.
Describe the general monitoring of thrombolytic agents
- aPPT - activated partial thromboplastin time
- CBC count
- Signs and symptoms of bleeding
- ECG
What is internal bleeding ?
Internal: intracranial, retroperitoneal sites, GI, genitourinary, or respiratory tracts
What is superficial bleeding?
Superficial: observed mainly at invaded or disturbed sites. Also including epistaxis (nose bleeding) and hematuria (blood cell in urine)
What are complications of thrombolytic therapy?
hemorrhage, allergic reactions, embolism, stroke, and repercussion arrhythmias, among others
What are some risk factors for hemorrhagic complications?
- Increasing age
- Lower body weight
- Elevated pulse pressure
- Uncontrolled hypertension
- Recent stroke or surgery
- Presence of a bleeding diathesis
- Severe congestive heart failure
