HC34+35: Secondary hemostasis I&II

Secondary hemostasis

Blood flows as a fluid through the blood vessels to all the organ systems. Upon injury, blood vessel loss has to be prevented. This is done via blood coagulation. Under normal conditions, blood coagulation is a tightly regulated process:

• Rapid formation of a clot upon injury
• Limited to the site of injury

Secondary hemostasis is the conversion of soluble fibrinogen to insoluble fibrin by thrombin (IIa):

• Strengthens the platelet plug
• Induces adherence and activation of cells involved in vascular repair
  • Fibroblasts
  • Smooth muscle cells

Thrombin plays a key role in the conversion of fibrinogen to fibrin. It sometimes also is called coagulation factor IIa.

Fibrinogen

Fibrinogen is also known as factor I and is very abundant in the plasma → normal levels are 1,5-3 gram/L. It consists of 2 symmetrical half-molecules, each consisting of 3 different polypeptide chains:

• Aα: linked together in the E-domain → the central parts
• Bβ: in the D-domain → the outer parts
• γ: in the D-domain → the outer parts

Formation of fibrin:

Fibrinogen is formed into fibrin as follows:

1. Thrombin cleaves away fibrinopeptides A and B → fibrin monomers are formed
   • Fibrin monomers are very sticky
2. 2 fibrin monomers stick together → dimers are formed
   • This is done via H-bonds
3. Coagulation factor XIIIa crosslinks different dimers → polymers are formed
   • Coagulation FXIIIa = transglutaminase
     • The enzyme factor XIII is converted into FXIIIa by thrombin (IIa)
   • The crosslinks are formed between the D-domains of the fibrin
   • These are covalent bonds

Thrombin

Thrombin is generated via the coagulation cascade. The coagulation cascade is a sequence of proteolytic reactions, which take place in parts of the surface of activated proteins. In each step, a pro-enzyme is converted to an enzyme, which activates the next pro-enzyme. These are slow reactions which can be sped up by co-factors → a kind of catalysts.

The coagulation cascade consists of 2 pathways:

• Extrinsic pathway: activation upon contact with tissue factor (TF)
  • TF was previously named coagulation factor III
  • The initiator is located outside the plasma
• Intrinsic pathway: activation upon contact with surfaces foreign to our body
  • E.g. glass → contact activation
  • Everything is located inside the plasma

The common pathway consists of the coagulation factors which play a part in both intrinsic and extrinsic pathways:

• FX
• FV
• FII

Extrinsic pathway:

There are several very important factors in the extrinsic pathway:

1. Tissue factor, factor VII
2. Factor X, factor V
3. Factor II: prothrombin

Tissue factor (TF) is a transmembrane glycoprotein and a non-enzymatic co-factor of FVIIa. TF is produced by fibroblasts and smooth muscle cells → is expressed on a "hemostatic envelope” around blood vessels. TF normally isn’t present in the blood compartment, but during pathological conditions it can be expressed:

• Inflammation: expression on endothelial cells and monocytes
  • This is called disseminated intravascular coagulation (DIC)
• Cancer: expression on tumor cells and circulating tumor-derived microparticles

The mechanism of the extrinsic pathway goes as follows:

1. Due to injury, tissue factor gets exposed in the blood
2. Small particles of active VIIa present in the blood bind to TF
3. Together, TF and VIIa bind to coagulation factor X → form Xa
4. Xa converts coagulation factor II to coagulation factor IIa (thrombin)
   • These reactions take place at a very slow rate
   • Xa is very essential for the formation of thrombin
5. The small amounts of IIa which get formed convert coagulation factor V to coagulation factor Va
6. Coagulation factor Va acts as a co-factor for the conversion of coagulation factor II to coagulation factor IIa
7. A large amount of thrombin (IIa) is formed

For this pathway to take place, additional components are necessary:

• Calcium
• Phospholipids

These components make the reaction a little bit faster. Only co-factor Va causes a real burst in the reaction. The actual function of Va is related to its shape.

Intrinsic pathway:

All elements of the intrinsic pathway are present in the blood plasma:

1. Factor XII, prekallikrein (PK), HMWK
2. Factor XI, HMWK
3. Factor IX, factor VII
4. Factor II (prothrombin)

The mechanism of the intrinsic pathway goes as follows:

1. A foreign substance makes contact with the blood
2. Due to unknown processes, factor XII binds to the foreign substance → converts coagulation factor XI into XIa
   • This is done with the help of prekallikrein (PK) and HMWK
3. XIa recognizes IX and activates it to IXa
   • This also is done with the help of HMWK
4. IXa activates X to Xa
5. Xa recognizes factor II and activates it to factor IIa (thrombin)
   • Factor IIa helps the conversion of coagulation factor V into coagulation factor Va → factor Va is a co-factor of the factor II to IIa conversion
   • Factor IIa also converts VIII to its active form VIIIa → VIIIa helps the conversion of X to Xa

Initiation and amplification:

The efficacy of coagulation can be measured with:

• Citrated blood
• Centrifugation
  • Of platelet-rich plasma
  • Of platelet-poor plasma

The prothrombin time (PT) and activated partial thromboplastin time (aPTT) are measured with citrated plasma:

• PT
  1. TF, calcium and phospholipids are added to citrated plasma
  2. Reactions occur
  3. The clotting time measured (in seconds) → the PT
  • This process is sensitive to shortage of FVII, FV, FII and fibrinogen → the extrinsic pathway
• aPTT
  1. An activator (such as glass powder), phospholipids and calcium are added to citrated plasma
  2. The clotting time is measured
  • This process is sensitive to shortage of FXII, PK, HMWK, FXI, FVIII, FX, FV, FII and fibrinogen → the intrinsic pathway

Thrombosis services use the “international normalized ratio” (INR). This ratio is derived from the PT, but is normalized to a standard. The PT and aPTT are abnormal in case of:

• Genetic deficiencies
• Liver disease
  • Most coagulation factors are produced in the liver
• Auto-immunity
• Disseminated intravascular coagulation
• Medication

Factor VIII:

Factor VIII cannot survive in the bloodstream without vWF → if there is no vWF, there is no factor VIII. vWF doesn’t play a role in the cascades themselves, but serves as a carrier of factor VIII.

Vitamin K dependent factors:

The biosynthesis of these proteins is dependent on sufficient levels of vitamin K in the liver:

1. Vitamin K is required for γ-coagulation of glutamic acid residues in the aminoterminus of the protein → 2 additional oxygen molecules are added to glutamic acid
2. The factors become negatively charged → can bind to calcium ions
   • FII, FVII, FIX, FX and protein C and protein S are vitamin K dependent
3. In the presence of calcium ions, the factors can bind to a negatively charged phospholipid surface
   • E.g. an activated platelet

Vitamin K is consumed during a reaction and later recycled. Lots of drugs interfere with this process:

1. Vitamin K antagonists cause vitamin K to become inefficient → coagulation factors aren’t sufficient
2. PT and aPTT go up

New alternatives for vitamin K antagonists are direct oral anticoagulants (DOACs):

• Direct IIa inhibitors
  • Dabigatran
• Direct Xa inhibitors
  • Rivaroxaban

These drugs are effective right away when taken orally, while vitamin K antagonists can take 5 days to become active.

Abnormal secondary hemostasis

Abnormal primary hemostasis has different symptoms than abnormal secondary hemostasis:

• Abnormal primary hemostasis
  • Petechiae, ecchymosis and spontaneous bleeding from mucosal surfaces
  • Increased bleeding time
  • Normal PT
  • Normal aPTT
    • Unless FVIII is affected by low vWF levels
• Abnormal secondary hemostasis
  • Single or multiple hematomas and bleeding into:
    • Subcutaneous tissue
    • Body cavities
    • Muscles
    • Joints
  • Normal bleeding time → platelet formation is normal → bleeding stops
  • Re-bleeding
  • Prolonged PT and/or aPTT

Deficiencies and inheritance:

Inheritance of abnormal secondary hemostasis deficiencies can be autosomal recessive or X-linked recessive:

• Autosomal recessive: more rare → 2 alleles need to be affected
  • FVII
  • FXI
  • FV, FX, FII
  • Fibrinogen
• X-linked recessive: less rare → males are dependent on 1 X-allele
  • FVIII → hemophilia A
  • FIX → hemophilia B

All these factors cause bleeding when a deficiency is present, although FXI doesn’t really cause bleeding → sometimes only gets detected during surgery. Lack of TF causes death during embryogenesis → not compatible with life.

Regulation of thrombin generation

Thrombin generation can be regulated via 3 processes:

• Initiation
  • Contact with tissue factor
  • Activation of all pro-enzymes
• Amplification
  • Feedback activation of the pro-co-factors FV and FVIII
  • Feedback activation of factor XI
• Inhibition
  • Inhibition of the enzymes
  • Inhibition of the co-factors

Inhibition of the enzymes:

Inhibition of the enzymes can be done with:

• Tissue factor pathway inhibitor (TFPI): suppresses the initiation of thrombin generation
  1. TFPI inhibits Xa while generating an Xa-TFPI complex
  2. The Xa-TFPI complex inhibits the TF-VIIa complex
• Antithrombin III (ATIII): anti-thrombin prevents diffusion of active coagulation enzymes
  • A serine protease inhibitor
  • Inhibits thrombin (IIa), Xa, IXa and XIa → ATIII kills these enzymes immediately
  • Action heparins can accelerate the inhibition 1000-fold
    • Heparins bind to ATIII when injected → changes its form and becomes extra effective
    • Used in case coagulation needs to be tempered
      • E.g. during surgery
    • There are 2 kinds of new generation heparins: induce a conformational change on ATIII but do not bind to IIa (thrombin), which old generation heparins do
      • Low molecular weight heparins: can be injected subcutaneously
      • Pentasaccharide heparins

Inhibition of the co-factors:

The co-factors FVa and FVIIIa can be inhibited with:

• Thrombomodulin
  • Present on normal endothelium as a transmembrane protein
  • A receptor and co-factor for thrombin
• Protein C
  • A pro-enzyme (serine kinase)
  • A vitamin K dependent protein
• Protein S
  • A non-enzymatic co-factor for activated protein C (APC)
  • A vitamin K dependent protein

When thrombin is bound to thrombomodulin, it undergoes a change in which it can convert protein C to activated protein C (APC). Together with protein S, APC inactivates factor Va and VIIIa → coagulation is shut down.

Bleeding and thrombosis

There is a narrow balance between pro- and anticoagulation:

• When there is too much pro-coagulation, there is a higher chance of thrombosis
• When there is too much anticoagulation, there is a higher chance of bleeding
• When there is too little pro-coagulation, there is a higher chance of bleeding
• When there is too little anticoagulation, there is a higher chance of thrombosis

There are 2 types of thrombosis:

• Arterial thrombosis
  • Caused by atherosclerosis
• Venous thrombosis
  • Caused by coagulation

Venous thrombosis:

There are different presentations of venous thrombosis:

• Deep vein thrombosis (DVT) in the legs
  • Not fatal
• Pulmonary embolism (PE) in the lungs
  • Fatal

Venous thrombosis can occur on different locations, but is most common on the legs. Both genetic and environmental factors are involved in the development. Genetic factors are:

• Deficiencies of natural anticoagulants
  • Deficiencies
    • Antithrombin deficiency: population frequency of 0,2%
    • Protein C deficiency: population frequency of 0,3%
    • Protein S deficiency: population frequency of 0,05%
  • Low prevalence
  • High relative risk of venous thrombotic events (VTE)
  • Loss-of-function mutations
  • Different mutations occur in different families
  • Mostly partial deficiencies
    • 1 allele is gone
    • Survival with 2 alleles gone isn’t possible
• Deficiencies of procoagulant factors
  • Factor V G1691A (FV Leiden) mutation: population frequency of 3-16%
    • Important in the coagulation cascade
    • Thrombin cleaves activation arginines → activates FV Leiden to Va
    • APC cleaves inactivation arginines → inactivates FV Leiden
      • An SNP in exon 10 causes an inactivation arginine to be a glutamine → less efficiently converted by APC → reduced rate of inactivation of FV Leiden
        • FVa Leiden is actually a better co-factor → gain of function mutation
        • High population frequency
          • Possibly was advantageous during the plague
  • Prothrombin G20210A mutation: population frequency of 3%
    • A G is switched to an extra A in the thrombin mRNA → the mRNA is more stable → more protein is formed → increased plasma prothrombin levels → coagulation is more active
      • The more stable the mRNA, the more protein can be formed
    • A gain of function mutation

Heterozygous cases of these mutations are mild, but homozygous cases are moderate or very severe.

Important risk factors:

Important risk factors for venous thrombosis are:

• Genetic risk factors
  • Venous thrombosis is a multifactorial disease
  • In many families there is a PC deficiency as well as a FV Leiden deficiency → increased risk
• Non-genetic risk factors
  • Age
  • Surgery
  • Trauma
  • Malignancies
  • Obesity
  • Hyperthyroidism
  • Oral contraceptives
  • Hormone substitution
  • Pregnancy
  • Puerperium
  • (Air) travel → reduced blood flow
  • Immobilization → reduced blood flow
  • Previous thrombosis