HC32+33: Primary hemostasis

Cancer and thrombosis

Thrombophlebtis is thrombosis due to infected vessels. Thrombotic events are the second leading cause of death in cancer patients, after death from cancer itself. The Trousseau syndrome describes the presence of thrombosis in relationship to cancer. Fibrin clots may be induced by cancerous processes:

• Cancer has an influence on coagulation → influences thrombosis
• Coagulation may have an influence on how the cancer will develop
• Thrombosis also has an effect on developing cancer.

Risk:

Patients with cancer have an increased risk for developing venous thrombosis:

• Odds ratio: 7
• Incidence: 24/1000/year
• Risk and time since diagnosis of cancer
  • 0-3 months: odds ratio of 54
  • 3-12 months: odds ratio of 14
  • 12-36 months: odds ratio of 4

The Khorana risk score predicts the risk of cancer associated thrombosis. This risk differs per cancer type and patient:

• Site of cancer
  • Very high risk → 2
    • Stomach cancer
    • Pancreas cancer
  • High risk → 1
    • Lung cancer
    • Lymphomas
    • Gynaecological cancer
    • Bladder cancer
    • Testicular cancer
• Prechemotherapy platelet count >350 x 10⁹/L → 1
• Hemoglobin level <10 g/dL or use of red cell growth factors → 1
• Prechemotherapy leukocyte count >11 x 10⁹/L → 1
• Body mass index >35 kg/m²→ 1

These scores can be added to predict the risk:

• >3 → high risk
• 1 or 2 → intermediate risk
• 0 → low risk

Patients with thrombosis also have a risk of developing cancer:

• Idiopathic (unprovoked) venous thrombosis: 10% of patients develop cancer in the next 3 years
  • Unprovoked: thrombosis with no other clear cause
• Provoked venous thrombosis: 1,6% of patients develop cancer in the next 3 years
• Single episode of superficial thrombosis: no clear association with cancer
  • If it returns, there is a connection

Mortality:

Cancer and thrombosis are a very bad combination. Extensive screening can identify occult tumors, but this does not improve the survival and can lead to false positive cases. Therefore, currently just a general screening is done.

Treatment of venous thrombosis:

In cancer patients, anticoagulation with low molecular weight heparin (LMWH) is more effective than vitamin K antagonists in prevention of recurrent venous thrombosis.

Older thrombosis studies also show a potential benefit of anticoagulant treatment. LMWH shows an improved 1-year survival only in patients with localized cancer. This is only a small advantage which isn’t proven. Even though there is a clear correlation, the conclusion is that there is no benefit of LMWH on cancer.

Mechanism of hemostasis

There are many factors which interfere with the coagulation system. As soon as the endothelium layer is broken or compromised, the coagulation begins:

1. First pathway: after the endothelium layer is compromised, vasoconstriction leads to a reduced blood flow
2. Second pathway: the blood gets into contact with subendothelial collagen → platelet adhesion and aggregation
   • A platelet plug is made
   • Serotonin and TxA2 are secreted → enhance vasoconstriction
3. Third pathway: exposition to tissue factor (TF) causes blood coagulation → cascade of enzymatic reactions → fibrin production
   • A network of fibers which create a network around the platelet plug
4. Thrombin is also formed → stimulates platelet adhesion and aggregation
5. Blood coagulation takes place on the surface of platelet aggregation

These processes together form hemostasis, which is followed by fibrinolysis → the platelet plug falls apart and the tissue is recovered.

Primary and secondary hemostasis:

This process can be divided into primary and secondary hemostasis:

• Primary hemostasis: closing the hole in the vessel wall fast
  • Consists of the first 2 pathways
    • Vasoconstriction leading to blood flow reduction
    • Platelet adhesion and aggregation making a platelet plug
• Secondary hemostasis: making a thick clot which will not break
  • Consists of the third pathway
    • Blood coagulation leading to fibrin production

There is a balance between bleeding and thrombosis which can be interrupted:

• If a patient lacks procoagulant factors → bleeding
  • Ecchymosis, hematoma, petechia, mucosal bleeding, hemathrosis, muscle hematoma, subconjunctival bleeding, mucosal bleeding, etc.
• If a patient lacks anticoagulant factors → thrombosis
  • Embolism, infarction, DVT, etc.

Primary hemostasis

Primary hemostasis makes it possible to close holes in endothelium fast. When the endothelium is compromised, platelets do 2 things:

• Bind to the collagen of the underlying tissue → adhesion
• Bind to each other → aggregation

Adhesion:

Adhesion forms a platelet plug by sticking platelets to collagen. There are 3 receptors on platelets which are involved with adhesion:

• GPVI: glycoprotein VI
  • Binds to subendothelial collagen
  • Always present on the surface of platelets, waiting until they see collagen
• GPIa: glycoprotein Ia
  • Binds to subendothelial collagen
  • Always present on the surface of platelets, waiting until they see collagen
• GPIb: glycoprotein Ib
  • vWF binds collagen to GPIb, together with factor VIII (FVIII)
    • vWF = von Willebrand factor
    • vWF can only do this when GPIb and FVIII gave been activated by collagen itself
    • FVIII: always sticks to vWF
  • Always present on the surface of platelets, but needs vWF to bind to collagen

Aggregation:

Platelet aggregation is the binding of platelets to platelets:

1. After adhesion, the morphology of platelets changes and they become activated
   • Platelets get a larger surface → can recognize each other more easily
2. The GPIIb/IIIa receptor is exposed on the surface of platelets and binds fibrinogen and vWF → platelets stick together
   • Fibrinogen and vWF are always present in the blood, but can only play their part in the cascade when the GPIIb/IIIa receptors are exposed and in their activated form
3. Platelets are activated → release certain proteins
   • α-granules
     • vWF
     • Fibrinogen
       • Also is present in the blood, but now has an increased release
   • Dense granules
     • Serotonin
     • ADP
   • Thromboxane A2(TxA2)
     • Cyclo-oxygenase converts arachidonic acids into TxA2
     • Induces vasoconstriction
     • Activates other platelets

Activation:

Through 3 receptors, activated platelets start showing the aggregation receptor:

• ADP receptor/P2Y12
  • When ADP is released, it binds to receptors of other platelets → get activated and start exposing their GPIIb/IIIa molecules → ADP is also released
• TxA2R
  • Thromboxane activates other platelets cells by binding to thromboxane A2 receptors
• Thrombin receptor/PAR1 (protease-activated receptor)
  • Thrombin is produced during secondary hemostasis
  • Stimulates platelet activation as well

In short, platelets that aren’t bound to collagen are also activated via these receptors.

Endothelium in hemostasis

As long as endothelium is intact, there will be no blood clots. Endothelial cells form a barrier between blood and tissue:

• No contact of blood with collagen
• No contact of blood with tissue factors

Intact endothelium prevents:

• Adhesion and aggregation of platelets
• Initiation of coagulation

Endothelium also has biochemical anti-thrombotic properties:

• Vasodilatation
• Inhibition of aggregation
  • Nitric oxide (NO)
  • Prostacyclin (PGI2)
    • Can bind to platelets and inhibit them
• Inhibition of coagulation
  • Thrombomodulin
  • Heparin-like glycosaminoglycans
  • Tissue factor pathway inhibitor (TFPI)
• Activation of fibrinolysis
  • Tissue plasminogen activator (tPA)
    • Tissue plasminogen activator

Disturbed primary hemostasis

Platelets:

Platelets are a main player in primary hemostasis. They are cell fragments of megakaryocytes, produced in the bone marrow. Their life span is about 7-10 days and the normal platelet count is 150-140 x 10⁹/L.

Thrombocytopenia:

Thrombocytopenia is characterized by low platelet blood counts → <30-50 x 10⁹. This causes an increased risk of bleeding. Causes of thrombocytopenia can be:

• Reduced production
  • Leukemia
  • Aplastic anemia
  • Chemotherapy
• Increased clearance/consumption
  • Immune thrombocytopenic purpura (ITP)
    Disseminated intravascular coagulation (DIC)
• Loss or bleeding

Thrombocytopathia:

Thrombocytopathia is characterized by platelet defects. Causes are:

• Congenital platelet defects
• Acquired platelet defects
• Medication: platelet aggregation inhibitors
  • Can reduce the risk of thrombosis

Congenital defects are:

• Bernard-Soulier syndrome
  • Impaired adhesion
  • Autosomal recessive
  • A rare disease
  • The GPIb receptor is not functioning → vWF cannot adhere to GPIb anymore → a large part of the adhesion stimulus is missing → platelets cannot bind to subendothelial collagen
• Glanzmann thrombasthenia
  • Defect in aggregation
  • Autosomal recessive
  • A rare disease
  • The GPIIb/IIIa receptor is not functioning → platelets can still adhere to the exposed collagen of the blood vessel, but links between platelets cannot be formed → fibrinogen and vWF cannot adhere → no platelet plug can be formed
    • The GPIIb/IIIa receptor is the only receptor which plays a role with aggregation of the platelets
• Storage pool disease
  • Reduction in secretion of granules or the number/content of granules in platelets → impairs the further activation of platelets in the blood
    • In α-granules which produce vWF and fibrinogen
    • In dense granules producing serotonin and ADP
  • A less rare disease
• Receptor defects
  • In the ADP receptor (P2Y12)
  • In the thromboxane A2-receptor
  • In collagen receptors (GPVI and GPIa)

Acquired defects are:

• Kidney failure
  • Very high levels of uremia causes platelet dysfunction
  • Platelets have less receptors due to an unknown mechanism
  • Characterized by bleedings
• Liver cirrhosis
  • Decline in function of the liver → substances are present in the blood which are normally are metabolized in the liver
  • Influence platelet function and decrease in platelet numbers
  • Unknown mechanism

Medication can also cause platelet defects:

• Inhibition of the conversion of arachidonic acid into thromboxane A2(TxA2) by cyclo-oxygenase → prevents activation of platelets
  • Aspirin
    • Irreversible inhibition → aspirin never gets off the platelets
    • Platelets only live 7-10 days → the effect fades after days
    • Aspirin is often used to stop thrombosis
  • NSAIDs
    • Reversible inhibition → is cleared in 24-48 hours
    • Adverse effect
• Blocking of the ADP receptor → platelets cannot be activated
  • Clopidogrel: irreversible
  • Prasugrel: irreversible
  • Ticagrelor: reversible
• Inhibition of the GPIIa/IIIb receptor → prevents aggregation
  • By monoclonal antibodies such as abciximab
  • The only way for aggregation is blocked → only given in very specific situations
  • Very high risk of bleedings

If the ADP route is blocked, the thromboxate route remains. If both routes are inhibited, the effect is very strong → after a myocardial infarction, 2 drugs are often combined.

Von Willebrand factors

Von Willebrand factors (vWF) normally circulate in the plasma and are produced by endothelium and megakaryocytes/platelets:

• Endothelium
  • Constitutive secretion
  • Regulated secretion
    • Weibel-Palade bodies are organelles which store vWF
      • Can secrete extra vWF when necessary
• Megakaryocytes/platelets
  • Regulated secretion
    • α-granules are organelles which store vWF

Secretion:

A vWF is a protein of many peptides. It isn’t a single protein, but first has to be linked together:

1. A dimer is formed in the ER
2. Dimers move to the Golgi-apparate → are linked together to form multimers
   • Multimers are very long molecules
3. The Golgi-apparate secretes Weibel-Palade bodies with vWF folded inside it
4. Weibel-Palade bodies enter the circulation and release the vWF → vWF unfolds again
5. When released into the circulation, ADAMT513 proteolyzes long vWF strands into shorter strands
   • ADAMT513 prevents that vWF forms too big aggregates with platelets
6. Different sizes of vWF are present in the plasma
   • HMW: high molecular weight multimers
     • Are the best at binding to collagen and platelet receptors
       • Have multiple binding sites
     • Factor VIII needs vWF to survive in the bloodstream
       • The size of vWF does not matter
   • LMW: low molecular weight multimers

Von Willebrand disease:

Von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure or function of vWF. It has a classification consisting of 3 classes:

• Type 1: partial quantitative deficiency of vWF
  • There is less vWF, which is functioning normally
  • Can have 2 causes
    • A null allele is inherited from 1 parent → less protein production
      • A null allele is a gene which is completely deleted or has a stop codon
    • A missense mutation
      • A single change of an amino acid which causes a mutant subunit that is incorporated in vWF
      • All multimers are abnormal → leads to the protein getting stuck in the cell or being removed from the circulation
        • Shorter half life
• Type 2: qualitative vWF variants
  • 2A: decreased platelet binding, deficiency of HMW vWF multimers
    • Because the lack of HMW vWF, the gap between subendothelial collagen and GPIb cannot be bridged and causes less platelet binding
  • 2B: increased affinity for platelet glycoprotein Ib (GPIb)
    • Even though there is no damage to the endothelium, vWF and FVIII bind to the GPIb receptor → the platelets to which these complexes bind are removed from the circulation → less platelets in the blood
  • 2M: decreased platelet binding receptors, no loss of HMW vWF multimers
    • Something is wrong in the binding of vWF to GPIb receptors → reduced binding of platelets → adhesion problems
  • 2N: decreased affinity for factor VIII
    • Normal platelet function, but a reduced affinity of factor VIII in vWF
    • Resembles hemophilia A
• Type 3: virtually complete deficiency of vWF
  • No factor VIII, because it needs vWF to survive in the bloodstream
  • A null allele is inherited from both parents → no protein production

Diagnostic tests of primary hemostasis

Screening tests:

The screening tests which are currently available are not very good. There are several kinds of possible screening tests:

• Platelet count
• Bleeding time
  • Tests the whole function of primary hemostasis
  • 2 incisions are made in the lower arm and pressure is put on the lower arm cuff → the time until the bleeding stops is measured
  • Variable if different technicians do the test
• Platelet function analyzer PFA100
  • Measures the “closure time”
    • The time until the hole is closed
  • An in vivo collagen membrane is made → blood is drawn from the patients and put into the machine → when blood touches collagen, platelets are made → platelets close the hole
  • A combination of the number and function of platelets and vWF is measured
  • Variable results

Specific tests:

Specific tests for primary hemostasis are:

• vWF concentration
• vWF function
• Platelet aggregation

Light transmission aggregometry is a way to measure platelet aggregation:

1. Blood is centrifuged a little → platelets will still be inside the plasma
2. Something that activates platelets, such as ADP or fibrinogen, is added
3. The plasma is put under a spectrometer → the difference in light transmission becomes visible → the more aggregation, the more light is let through
   • Normal situations: when activated with ADP, platelets will excrete more ADP activating more platelets
     • If ADP is added, the light transmission increases
   • Storage pool deficiency: there is no ADP present in platelets → no other platelets are activated
     • The second wave is missing
   • Glanzmann: there are no functional IIb/IIIa receptors → ADP cannot bind to these receptors → ADP doesn’t do anything
     • No aggregation at all
     • Flat light
     • Ristocetin can artificially activate vWF
       • Only GPIb receptors cause platelets to start to stick together
   • Bernard-Soulier: there are IIb/IIIa receptors, but no Ib receptor → reaction to ADP but not to ristocetin because vWF cannot bind