Lecture 20 Thrombotic Conditions and Laboratory Assessment

Question 1

What is a thrombus?

Answer 1

Thrombus is an abnormal formation of a platelet or fibrin clot obstruction of a blood vessel

Question 2

What are some causes of a thrombus?

Answer 2

Many causes:
- Circulatory stasis
- Abnormality in the coagulation system & coagulation control mechanisms
- Abnormal platelet function
- Blood vessel wall disruption
- Leukocyte activation

Question 3

Where does Venous Thromboembolic Disease (VTE) commonly occur and what are the symptoms?

Answer 3

1. Most commonly occurs in the veins of the calves and upper legs
2. Symptoms are localized pain, sensation of heat, redness and edema

Question 4

What more serious complications can occur in Venous Thromboembolic Disease (VTE)?

Answer 4

1. Fragments of the clots (thrombi) may separate and move quickly to chambers of the heart, the brain and lodge in the lung called pulmonary emboli (PE)
2. These blockages cause ischemia (decreased blood flow)and necrosis of tissue behind the block causing heart attack, stroke, and impaired breathing

10% to 15% of PE patients die within 3 mos.

Question 5

What is Arterial Thrombosis?

Answer 5

Arthrosclerotic plaques caused by accumulation of lipoproteins mainly LDL

Question 6

Describe the mechanism/process by which Arterial Thrombosis occurs?

Answer 6

1. Activated platelets, monocytes and macrophages embed fatty plaques suppress nitric oxide a vascular relaxing factor
2. Small plaques rupture occluding arteries and exposing collagen and releasing Tissue factor
3. Platelets binding to vWF will adhere to collagen, then aggregate forming platelet plug
4. Tissue Factor combined with Factor VIIa will initiate the coagulation cascade causing a fibrin clot

Question 7

What patients are frequently tested for Hypercoaguable states?

Answer 7

1) Venous or arterial thrombosis.
2) Transient ischemic attacks (TIA’s)
3) Peripheral Vascular Disease
4) Personal or family history of thrombotic events.
5) Prior to surgery, pregnancy, oral contraceptive use, estrogen therapy, Decreased antithrombin and people with personal or family history of thrombosis.

Question 8

What is a natural anticoagulant in the body? What factors does it affect?

Answer 8

Antithrombin: Natural anticoagulant to activated forms of thrombin, Factors IX, X, XI and XII. Most important factors inhibited are IIa and Xa.

Question 9

What defects and disease are associated with hereditary deficiencies of antithrombin?

Answer 9

Hereditary deficiencies both quantitative and qualitative. D.I.C and Liver Disease also causes low levels

Question 10

What therapy can deplete levels of antithrombin if not monitored properly?

Answer 10

Heparin therapy over a sustained period can deplete AT causing increased risk of thrombosis.

Question 11

How is antithrombin typically measured?

Answer 11

Usually a chromogenic assay: Amount of chromophore released is proportional to AT concentration.

Question 12

What can give rise to heparin resistance?

Answer 12

Low antithrombin level can give rise to heparin resistance.

Note: Without adequate amounts of Antithrombin Heparin cannot work properly as an anticoagulant. If patients are not responding to Heparin therapy an Antithrombin level assay should be performed to rule out Antithrombin deficiency as the cause.

Question 13

What does Antithrombin require for effective anticoagulant activity?

Answer 13

Aka Heparin co-factor
Most important coagulation inhibitor
In vivo heparin sulfate (from endothelial cells and platelets) + commercial heparin
↑ AT activity

Need to confirm what “Aka Heparin …” means.

Question 14

Where is antithrombin made?

Answer 14

Synthesized in the liver

Question 15

What factors does anti-thrombin inhibit?

Answer 15

Naturally inhibits coagulation by forming complexes with factors II, IX, X, XI, XII therefore neutralizing them

See slide 15 for a list of all of AT’s functions.

Question 16

In what conditions is anti-thrombin deficient in?

Answer 16

Deficiencies in AT such as those conditions listed below can lead to severe, even fatal thrombotic events:

1. Hereditary seen in 1:2000 to 1:5000 of the general population
2. DIC
3. Liver disease
4. Nephrotic syndrome

Question 17

What are Protein C and S?

Answer 17

Protein S is a cofactor to Protein C
When complexed, Protein C and S are potent inhibitors of Va and VIIIa

Question 18

What is the impact of even an heterozygous deficiency in either S or C proteins?

Answer 18

Heterozygous deficiencies lead to a 2-10 fold increased risk of thrombosis

Deficiencies result in serious thrombotic disorders.

Question 19

What vitamin is protein C and S dependent on?

Answer 19

Vitamin K dependent (like II, VII, IX, and X)

Question 20

How does protein C and S degrade Va and VIIIa?

Answer 20

1. Thrombin binds to endothelial receptor thrombomodulin
2. Thrombin + thrombomodulin cleave Protein C to form activated Protein C (APC)
3. Protein S complexes to activated Protein C (APC) which degrade Va and VIIIa

Question 21

What is Factor V Leiden?

Answer 21

Factor V Leiden is a genetic mutation seen in 3-8% of Northern European Caucausians causing the Factor V molecule to be resistant to Activated Protein C causing a 3-18 fold increased risk of thrombosis.

Question 22

What does the Activated Protein C Resistance test do?

Answer 22

Activated Protein C Resistance: test for Factor V Leiden Mutation which provides resistance of Factor V to Activated Protein C. Abnormalities should be confirmed by DNA testing.

Question 23

What is looked at in Factor V Leiden DNA testing?

Answer 23

Factor V Leiden DNA testing: describes as single point mutation which provides the resistance to APC.
● DNA testing important to confirm heterozygous or homozygous state.

Question 24

What test discovers a gene defect that leads to increased amounts of Prothrombin and increased risk of thrombosis?

Answer 24

Prothrombin Mutation Test (G20210A)
● Is also a gene defect which results to increased amounts of Prothrombin and increased risk of thrombosis.
● Presence detected by DNA testing.

Question 25

How does increased levels of Homocysteine increase risk of thrombosis?

Answer 25

Hyperhomocysteinemia Prothombotic effects: ● Decrease of endothelial cell Tissue Plasminogen Activator (TPA) binding sites. ● Activation of Factor VIIa and V ● Inhibition of protein C and heparin sulfate ● Increased prothrombin fragments ● Increased blood viscosity ● Decreased antithrombotic activity due to changes in thrombomodulin function

Question 26

How does oral contraceptives increase risk of thrombosis?

Answer 26

1. Due to acquired resistance to activated Protein C 2. Increase in factors II and VII and fibrinogen Note: Increase risk especially in people with other conditions such as hereditary defects; i.e. Factor V Leiden, Protein C and S deficiencies etc.

Question 27

What is the name of a group of inhibitors that bind with protein-phospholipid complexes and sometimes called non-specific inhibitors?

Answer 27

Lupus Inhibitors (anticoagulant): Lupus inhibitors / antibodies produced in response to viral infections, drugs, tumor necrosis factor, autoimmune inflammatory disorders are a group of immunoglobulins which bind to a variety of protein-phospholipid complexes and are sometimes called non-specific inhibitors These antibodies alter the phospholipid surface as described above.

Question 28

What does Lupus Inhibitors (anticoagulant) inhibit?

Answer 28

1. Inhibition of fibrinolytic system (factors XII and prekallikrein) 2. Inhibition of prostacyclin release from enthothelial cells. (inhibitor of platelet aggregation) 3. Inhibition of Protein C and S systems. 4. Inhibition of Annexin V (significant role in the down regulation of extrinsic (tissue) pathway 5. Inhibition of beta-2-glycoprotein I (acts with protein S) Note: Scientist continue to investigate how lupus inhibitors cause thrombosis

Question 29

What are the effect of Lupus inhibitors on coagulation tests?

Answer 29

Lupus inhibitors cause an elevation in coagulation tests due to interference with protein/phospholipid reactions in the coagulation cascade .

Question 30

What is the purpose of Antiphospholipid Testing and what condition is it found in?

Answer 30

Demonstrates presence of antibodies to phospholipid (negatively charged lipids), commonly found in System Lupus Erythematosis (SLE).

Question 31

What kind of antibodies can be found in Antiphospholipid Testing?

Answer 31

Antibodies can be IgM, IgG, IgA

Question 32

What conditions are associated with positive Antiphospholipid Tests?

Answer 32

Positive tests are strongly linked to conditions of venous, arterial thrombosis, recurrent fetal loss, thrombocytopenia, cerebral infarction.

Question 33

What is Heparin Co-Factor II?

Answer 33

Heparin Co-Factor II Inhibits thrombin Less affinity and reacts more slowly than AT

Question 34

What does Alpha 2 Macroglobulin inhibit?

Answer 34

Alpha 2 Macroglobulin Inhibits thrombin and Kallikrein Slower than AT

Question 35

What factor does Alpha 1 Antitrypsin inhibit?

Answer 35

Alpha 1 Antitrypsin: Alpha globulin inhibits FXIa Inactivates thrombin at a weak rate

Question 36

What is the main inactivator of XIIa, XIa, and Kallikrien contact factors?

Answer 36

C1 inactivator Main inactivator of XIIa, Xia, and Kallikrien contact factors

Question 37

What is the principal regulator of the tissue factor pathway? Where is it synthesized?

Answer 37

Tissue Factor Pathway Inhibitor (TFPI): Synthesized by endothelial cells Principle regulator of the tissue factor pathway Remember . . . . in vivo TF:VIIa activates IX and X

Question 38

How does TFPI prevent activation of X and IX?

Answer 38

TFPI binds and inactivates Xa TFPI:Xa complex will then bind to TF:VIIa and prevent activation of X and IX

Question 39

List three anticoagulant therapy drugs and what they inhibit?

Answer 39

1. Aspirin Inhibits platelet aggregation 2. Heparin (unfractionated) Inhibits factors II, IX, X, XI, XII 3. Warfarin Inhibits factors II, VII, IX, X

Question 40

How does aspirin work as an anticoagulant to treat thrombotic events? How long does it last?

Answer 40

Aspirin Anticoagulant Therapy 1. Aspirin inactiviates cyclooxygenase Blocking thromboxane A2 production 2. Interferes with platelet aggregation 3. Irreversible - Lasts the rest of the platelets life, ~7-14 days Can be used to treat thrombotic events

Question 41

What does aspirin prolong?

Answer 41

Most common drug associated with prolonged Bleeding Time.

Question 42

What does Heparin Therapy (Unfractionated) inhibit?

Answer 42

Inhibits intrinsic and common factors 1. Thrombin 2. IX 3. X 4. XI 5. XII

Question 43

How can you measure heparin therapeutic range? What is the range?

Answer 43

Measured by APTT Test Therapeutic range: 0.2 – 0.4 units/mL Unfractionated Heparin

Question 44

What does heparin enhance?

Answer 44

Anti-thrombin enhancer 1. Heparin’s action on these factors (X11a, X1a, IXa, Xa, VIIa, Va, VIIIa, Thrombin) is mediated through the action of anti-thrombin (speeds up AT) 2. Exposes more of its active sites so they can bind to the activated coagulation factors more efficiently and render them ineffective. 3. Heparin increases ATIII effect several thousand times fold !!! ( 2 – 10,000 times fold). See slide 31 for diagram.

Question 45

What is heparin therapy the choice treatment of?

Answer 45

Choice for immediate treatment of: 1. Acute venous thrombosis 2. Pulmonary embolism (PE)

Question 46

How is heparin therapy administered?

Answer 46

Administered: 1. Intermittent bolus (concentrated) injection IV every 4-6 hours 2. Continuous IV infusion 3. Subcutaneous injection every 12 hours Dose too ↑ = bleeding; dose too ↓ = thrombosis

Question 47

What can cause heparin resistance?

Answer 47

Heparin Resistance Can be due to ↓ levels of anti-thrombin (<50%) and ↑ levels of stress factors I, V and VIII. Note: May require higher doses of Heparin to overcome their pro-coagulant effect

Question 48

What is the goal of the heparin anticoagulant therapeutic range?

Answer 48

Therapeutic range to provide proper anticoagulant effect and prevent bleeding should be established for each lab using present heparin stock

Question 49

What test is used to establish the therapeutic range for heaprin?

Answer 49

Test used to establish therapeutic range is heparin response curve. APTT times are performed on concentrations of heparinized plasma from 0.0 to 0.5 units of heparin/ml of plasma. Therapeutic range of Heparin Response curve is APTT times typically between 0.2 to 0.4 units/ml.

Question 50

How can too high of dose of heparin be reversed?

Answer 50

Too high doses of heparin can be reversed by administration of Protamine Sulfate

Question 51

What occurs in heparin induced thrombocytopenia (HIT)?

Answer 51

Heparin Induced Thrombocytopenia (HIT) HIT: condition where antibodies are produced against the heparin/platelet factor 4 complex on the surface of the platelet. This leads to aggregation of platelets, thrombocytopenia with arterial and venous thrombosis.

Question 52

What blood count should be performed prior to and during heparin therapy?

Answer 52

Platelet count should be performed prior to and during heparin therapy to detect Heparin Induced Thrombocytopenia (HIT).

Question 53

How does Heparin Induced Thrombocytopenia (HIT) lead to thrombocytopenia?

Answer 53

Aggregates cleared by spleen Leading to thrombocytopenia Note: Patients with HIT will require an alternate anticoagulant therapy

Question 54

What is Low Molecular Weight Heparin?

Answer 54

1. Produced from unfractionated heparin by chemical and enzymatic depolymerization = reduced size 2. Reduced activity to bind thrombin but increased ability to bind Xa 3. Effective antithrombotic agent especially in deep vein thrombosis (DVT).

Question 55

What are the advantages of LMWH over standard heparin?

Answer 55

1. Does not interact with platelets so less chance of bleeding. 2. LMWH shows little or no binding to plasma proteins providing greater bioavailability and more predictable clearance from the circulation than standard heparin. 3. Lower incidence of HIT. As a result laboratory monitoring is not necessary. 4. LMWH is the heparin of choice for out-patients because it can be given once or twice per day.

Question 56

If LMWH monitoring is necessary what assay should be performed?

Answer 56

In rare cases where LMWH monitoring is necessary it should be done with anti-Xa heparin assay, not the APTT.

Question 57

What is Warfarin?

Answer 57

Oral anticoagulant (can be given to people not requiring hospitalization). Used most commonly for treatment of venous thrombosis Brand name: Coumadin May also hear it as Coumarin

Question 58

When can Warfarin be given as an oral anticoagulant?

Answer 58

Warfarin should only be started when therapeutic range of heparin is achieved. 1. Heparin is faster acting 2. Stable warfarin anticoagulant effect is not achieved for several days (36-72hrs) to (72-96hrs) 3. Heparin is discontinued once stable anticoagulant activity from warfarin is achieved. There is usually a ≈5-7 day overlap.

Question 59

What does Warfarin interfere with and how do you reverse it?

Answer 59

Interferes with Vitamin K metabolism Reverse affects of warfarin by administering Vitamin K and Fresh Frozen Plasma.

Question 60

What vitamin K dependent proteins does Warfarin interfere with?

Answer 60

Vitamin K dependent proteins C – inhibits Va and VIIIa S – cofactor of C Z – cofactor to ZPI which inhibits Factor Xa

Question 61

What is Warfarin's effect of the rate of reduction and why is this important?

Answer 61

Warfarin’s effect on the rate of reduction is related to the metabolic half life of the affected factors. Protein C half-life is shorter than the half-life of any of the other factors that Warfarin inhibits. This is important because in the initial stages of Warfarin therapy patients are actually at an increased risk of thrombosis hence the need for a Heparin-Warfarin overlap period. Note: Factor VII: short half-life, decreases first 5-6 hrs Protein C: shorter half-life 4-6hrs (inhibits Factor Va and Factor VIIIa) Protein C decreases first, so patient is at greatest risk of thrombosis at the beginning of warfarin therapy Factor IX: 28-40 hrs Factor X: 40-50 hrs Factor II: 48-60 hrs.

Question 62

How long does it take for stable warfarin anticoagulation to be achieved?

Answer 62

Stable warfarin anticoagulant effect is not achieved for several days (36-72hrs) (72-96hrs)