HEMOSTASIS

All the physiological mechanisms to avoid bleeding and haemorrhages after a wound and at the same time to
maintain the blood fluidity into blood vessels.

1) PRIMARY HEMOSTASIS
Very rapid (seconds) formation of the primary haemostatic plug to block the bleeding in small vessels
(capillaries, venules). It mainly involves platelets;
Platelets derive from the fragmentation of the BM’s megakaryocytes, even if there is a pool of platelets
sequestered in the spleen.
Number of circulating platelets: 150.000-450.000/mL of blood.
Average lifespan in bloodstream: 2 weeks; diameter: 2-3 micrometres.
They don’t have nucleus (no gene transcription and protein synthesis)  as a consequence they need
components taking from other cells. Platelets are characterized by the open canalicular system, a tunnelling
network of surface-connected channels made by platelets’ PM  this surface presents protrusions and very
deep pockets to increase the surface in contact with the environment. Platelets contain granules that are:
a) Dense bodies or delta granules – low molecular weight proteins, electrolytes, calcium, ADP and ATP.
b) Alpha-granules – high molecular weight components, like growth factors, coagulation factors and other
regulatory molecules.

The formation of primary plug is divided into 3 sub-phases:

I) Platelet adhesion to the matrix – they interact mainly with collagen fibres through specific
receptors (glycoproteins) to form 2 big complexes:
 GpIa/ GpIIa complex mediates the direct binding of platelets to collagen; one single molecule of
collagen is bound by one single platelets.
 GpIb receptor mediates the binding of the collagen through the vWF. The vWF is a multimeric protein
constituted by different monomers (normally present in a globular configuration) and, when there is a
damage in the endothelium, the collagen is exposed and vWF can bind the collagen  this mechanical
stress induces the linearization of the wWF’s monomers. The exposure of multiple binding sites for each
monomer allows that multiple platelets can bind to a single collagen molecule.
Defects in GPI complex or in the vWF lead to haemorrhagic events  no binding to collagen.
vWF has also the function of binding the factor VIII in order to stabilize it  defects in vWF= defects in
the stabilization of coagulation factors.
vWF is produced by endothelial cells (stored in Weibel-Palade bodies) and megakaryocytes (stored in α-
granules).

II) Platelets release reaction – once collagen in bound by receptors, there is a transduction signalling
inside platelets that stimulates the activation of the cytoskeleton and their change of shape functional
for:
 the release of the content inside granules  all these high/low molecular weight molecules are able to
activate the secondary haemostasis and to support inflammation related to a wound. [In particular ADP
and thromboxane A2 are needed to stimulate platelets in the third phase].
 The exposure of phospholipid complex (FP3) on their membrane to activate the coagulation cascade;
Platelets themselves can activate the secondary haemostasis by binding the endothelial matrix.
III) Platelets aggregation – stimulated by ADP and thromboxane A2.
 ADP stimulation  the glycoprotein complex GpIIb/GpIIIa is activated by the binding of ADP and
it’s able to bind fibrinogen  formation of large platelets aggregates. This link between the complex and
the fibrinogen is stabilized by the thrombospondin (present in the dense bodies of platelets).
 Thromboxane A2 stimulation  this molecule is produced by the COX pathway activation.
NSAIDs therapy  block of COX  block of the TXA2 production  no platelets’ aggregation.
Indeed, these drugs are a prophylactic treatment to decrease the thrombotic risk.

2) SECONDARY HEMOSTASIS
It requires a longer time (minutes) and is important to strengthen the primary haemostatic plug ( formation
of the secondary haemostatic plug), through the activation of coagulation factors in order to form a non-
soluble fibrin, acting like a gel. It involves the stop bleeding from large vessels with high pressure.
The final step of the coagulation cascade is the activation of fibrinogen ( fibrin) activated by thrombin.

The coagulation cascade is divided into:

A) INTRINSIC PATHWAY
 Activation of factor XII (in presence of PK, HMWK, phospholipids exposed by platelets);
 Phospholipids + calcium  activation of factor XI;
 Factor XI in presence of factor VIII + calcium + membrane phospholipids  activation of factor X.
 Factor X in presence of factor V + phospholipids + calcium  activation of precursors of thrombin
and activated thrombin can activate fibrinogen  then transformed in fibrin.

B) EXTRINSIC PATHWAY
In this case the stimulus is the tissue damage – release of factors that normally are inside tissues (defined
as “thromboplastin” (mainly constituted by the tissue factor, a specific protein) responsible for the
activation of factor VII. Once factor VII is activated + calcium + tissue factor  activation of factor X
to continue the common pathway of the cascade.

N.B. The coagulation cascade is a multi-step process because in this way there can be:
 a specific control step (inhibition/stimulation of coagulation)
 an avoidance of complete blood clotting: we need that only a portion of fibrinogen in the bloodstream is
converted in fibrin, otherwise all the blood would clot.

All the coagulation factors are produced by liver and released in bloodstream. The half-life of these proteins
is very variable but the less long-living is the factor VII (6 hours only)  in case of acute liver injury, the
first factor to be missing is the VII (present in the extrinsic pathway, so altered before the intrinsic).

Control of coagulation
Two major processes:
o Activity of protein C and S, mediated by thrombomodulin (produced by endothelium). To inhibit the
coagulation, there is the formation of a dimer formed by C protein (catalytic) and S protein (structural).
This dimer is able to cleavage factor V and factor VIII  decrease of the local coagulation factors.
o Activity of antithrombin III, produced by liver and able to bind and inhibit factor Xa and thrombin.

One common anti-coagulant is heparin – able to stabilize antithrombin III and inhibit thrombin.
N.B. thrombomodulin  produced at a local level.
Antithrombin III  produced at a systemic level (in case of inflammation).

 In order to overcome the inhibitory effect of the antithrombin III, an huge amount of activated thrombin
is required.

To study coagulation in lab, 2 tests:

 Calcium chelating substances – EDTA (calcium to saturate the chelating agents and avoid coagulation).
 Thrombin inhibitors – to stabilize anti-thrombin and keeps the blood liquid. Not reversible.

Among the coagulation factors there are some vitamin K-dependent factors (factor II, VII, IX, X) that are
post-translationally modified in the liver before being released in the bloodstream and these modifications
require a carboxylase that is vitamin-K dependent (used as co-enzyme).
1) Activity of carboxylase able to add an additional carboxyl group in the protein structure of factors.
2) Then calcium interacts with these two carboxyl groups  conformational change  activation.

Ex: Warfarin is a dicumarol drug used to decrease the thrombotic risk due to their ability as vitamin K
competitive inhibitors, blocking the synthesis of new functional factors.
 These therapy needs to be continuously monitored: if the vitamin K by diet increases/decreases, we need
to increase/decrease the drug’s concentration.
 New oral anticoagulants: they are small molecules able to inhibit specific protein factors (like factor X).

FIBRINOLYSIS
This process is required after the secondary haemostasis to remove and dissolve the fibrin clot/thrombi.
It’s performed by plasmin, a protease that is generated by an inactive precursor (plasminogen, by liver).
Plasminogen can be activated to plasmin according to:
 An extrinsic pathway – triggered by t-PA (tissue plasminogen activator), secreted by endothelial cells.
 An intrinsic pathway – triggered by circulating urokinase (activated by XII, XI, HMWK, kallikrein),
that is a serine protease release by connective tissue’s cells (fibroblasts, epithelial cells and
macrophages) of renal tubes.

These pathways are also controlled by PAI (plasminogen activator inhibitor).

Fibrinolytic drugs are used to re-open vessels closed by clots – administration of recombinant urokinase or
recombinant PA.

Deficiency in the haemostatic process  bleeding, with different physical manifestations:

 Petechiae, very small haemorrhages (1-2 mm) at the capillary level.
 Purpura, bigger than petechiae (3 mm), very superficial – under the skin.
 Ecchymosis, larger bleeding (1-2 cm), red or blue at the beginning.
 Hematomas, depth in muscles, kidneys. Result of defect in secondary haemostasis (large vessels).
The bleeding can collect in some anatomical cavities, like pleura or pericardial cavity, in joints, nose,
mouth and so on. They can start, then stop and star again.
Lab tests to evaluate the primary haemostasis
a) Bleeding time – we need to perform different and constant wound on patients to measure the time
needed to arrest bleeding. It’s a standardized test, to maintain constant the pressure. Generally bleeding
stops within 6 minutes, if it takes longer (more than 10 min) there is a problem in the primary
haemostasis; by using paper to collect blood, we can see the decrease amount of blood during time.
b) Platelet count – in the hematocytometry test we have information about the number of platelets (normal
range: 150.000-400.000/microL); if this value decreases, we have bleeding. The increase of bleeding
time is proportional to the decrease in the number of platelets.
The clinical threshold is 50.000/microL  values below that are pathological.
c) Platelets aggregation test – if we don’t see any reduction in the number of platelets, this test is very
useful  we have to take platelets outside the body (after centrifugation, there is the extraction of
plasma located near the interface between plasma and cells rich in platelets – platelet-rich plasma) that
react with some reagents (that resemble collagen or directly ADP) to activate them – adhesion phase –
and favor their aggregation. When there is aggregation  decrease in turbidity during platelets
aggregation (evaluable by a photometer, the final value is compared to the normal value of a protein-rich
sample).

So the reagents to add can be different:

 ADP – important to favor the binding of platelets to each other through the interposition of fibrinogen.
In this case, if we have problems, they can be related to inherited diseases or to side effects of aspirin
treatments or other drugs blocking the TXA2 activity.
Glanzmann’s thrombasthenia – defects in genes encoding for components of the GpIIb/GpIIIa
glycoprotein complex.
Bernard Soulier syndrome – inherited defect of GpIb complex component (platelets receptor is defective)
 defective platelets adhesion.
Hypofibrinogenemia – lack in fibrinogen; altered response to ADP test. It’s mainly characterized by a
disseminated intravascular coagulation.1

 Ristocetin – antibiotic not anymore used due to its side effects and it’s limited sensitivity; it was able to
mimic the effects of collagen, mimicking the adhesion of the first sub-phase of the primary haemostasis.
This test is important in the diagnosis of von Willebrand disease – autosomal dominant disease with
incomplete penetrance, defect in the primary haemostasis and of Bernard Soulier syndrome.
Ristocetin + wWF defect  no aggregation.
Ristocetin + Gp1b defect  no aggregation.
Ristocetin + normal serum (wWF defect)  aggregation.
Ristocetin + normal serum (Gp1b defect)  no aggregation.
This test can be used for quantitative measures of vWF secretion – this is just a classification of primary
haemostasis disorders, then further analyses are required for the diagnosis.
Lab tests to evaluate the secondary haemostasis
1
Very potentially severe syndrome, mainly caused by: 1) systemic activation of the haemostatic process (thrombotic
events) and 2) the consumption of platelets and coagulation factors. Very difficult to recognize. DIC is due to:
- the intrinsic pathway (widespread or diffuse endothelial damage)  heart immune disorders, infections, shock.
- the extrinsic pathway (diffusion of tissue factors in bloodstream)  obstetric accidents, neoplasms, sepsis from
gram- bacteria (inhibition of thrombomodulin release by endothelium), infections, tissue damage (burns).
- both intrinsic and extrinsic pathways  infections, burns, shock, snake poisoning, acute pancreatitis.
Clinical classification: acute (massive and frequent haemorrhages), subacute (thrombosis of microcirculation or
minor bleeding), chronic (silent aspect, only evaluable by lab data analysis).
The main approach is evaluating the time leading to the formation of clot (naked eye observed or measured
by automatic machines) after the addition of different specific agents to plasma. The sample is uncalcified
plasma, with coagulation factors, to evaluate intrinsic or extrinsic pathways.

 aPTT: activated partial thromboplastin time – to evaluate intrinsic and common pathways by using a
granulated activating agent, the kaolin coated by phospholipids to increase the surface of contact + Ca2+.
In this case we are adding the partial thromboplastin (purification of a tissue homogenate).
We need to recalcificate the plasma to measure the time until the clot formation.
This aPPT is expressed in seconds and can vary from 28s to 40s. It can also be expressed as a ratio
between the time of normal plasma/time of the patient plasma  if the ratio=1 (normal sample), if the
ratio=2 (twice the time of normal plasma).
 PT: prothrombin time (or quick time) – to evaluate the extrinsic and common pathways by adding the
full thromboplastin (tissue factors derived by animal tissues) + calcium. After putting the solution at
37°C (same for aPPT) we can start measuring the time. It is shorter than aPTT, generally 11-13 seconds
and is also expressed as a ratio.

Haemophilia (X-linked recessive)

Mutations in factor VIII – Haemophilia A (more frequent)
Mutations in factor IX – Haemophilia B (rarer)

Defects in the secondary haemostasis  bleeding in large vessels: hematomas and hemarthroses (bleeding
in the joint cavity) or internal bleeding.
Different degrees of severity according to the % of coagulation factors (totally absent of present in some %):
- > 5%: very mild disorder
- < 5%: bleeding, even spontaneous once.
- < 1%: severe haemophilia; repeated bleeding episodes.
- 1 < % < 5 %: bleeding but less frequently.

Diagnosis
First of all we need to explore the intrinsic pathway: aPTT test – factor VIII and IX are both involved in the
intrinsic pathway and encoded by genes present in the X-chromosome.
To classify haemophilia: complementation test  by adding factors we evaluate the blood ability to restore
the coagulation.
- if we add factor VIII to plasma and there is the rescue: haemophilia B (no mutation in factor VIII).
- Activation via intrinsic (kaolin + phospholipids + calcium):
o In case of haemophilia A  prolonged aPTT;
o In case haemophilia B  prolonged aPTT;

If in the same experiment, we add also plasma from a patient with haemophilia A:
o In case of haemophilia A  prolonged aPTT
o In case of haemophilia B  normal aPTT
Lab tests to evaluate the efficiency of a treatment
- Measure the PT (prothrombin time) to evaluate the extrinsic pathway. The PT value can be normalized
with the INR system (international normalized ratio) that takes into account the activity of
thromboplastin used: there are different thromboplastins with different reference value, like ISI
(international sensitivity index), that is provided by sellers and introduced in the data sheet.
 INR is PT (of patient)/ PT (of control) to the potency of ISI.
 INR depends on the reagent and the activity of the thromboplastin used (range from 1, 2, 3 etc).
 PT is not a very good test to see the hyperactivity of the thrombotic event but is very good to measure
the defects of the protein transcription.
If we use less active thromboplastin – lengthening of the times in defective plasma (extrinsic pathway);
If we use a more active thromboplastin – shortening of the times in defective plasma (extrinsic pathway).

New drugs, the NOACs (new oral anticoagulants) – inhibitors of thrombin or factor Xa. This is a
prophylactic treatment in patients with atrial fibrillation or artificial cardiac valves and the efficacy of the
treatment is monitored usually by PT test.

Lab tests for the assessment of hypercoagulability state

They are able to assess the thrombotic risk (thrombophilia) – increased tendency in developing thrombotic
events, mainly venous (legs or central venous thrombosis). 5 group of tests:
a) Activity of natural anticoagulants antithrombin III, protein C and S – there are some inherited conditions
in which these factors are lower expressed (autosomal dominant disorders, homozygosity would not be
compatible with life).

b) Mutations in factor V  factor under the control of protein C and S, that can degrade it very quickly.
In these pathological conditions, due to a specific aminoacidic composition, factor V becomes resistant
to the activity of this complex. More frequent in north Europe.

c) Mutations in PT gene  patients with higher levels of PT have a specific post-transcriptional mutation.
More frequent in south Europe.

d) Blood levels of homocysteine  in case of mutation there is an increased level of homocysteine in

blood, with a toxic effect on endothelium, so this has a double effect:
o Damage in tissues – increase in the thrombotic risk
o Action on LDL, increasing the level of atherosclerosis and so thrombosis.

e) Antiphospholipid Abs  this acquired condition is very common in different autoimmune disorders;
these Abs can increase the thrombotic risk. But these events are developing in a specific time, possibly
due to other risk factors (smoking, contraceptive drugs, surgery etc).

Lab tests for the assessment of fibrin formation/degradation

- Determination of fibrinopeptides A and B
Fibrinogen is composed by 3 chains (alpha, beta and gamma) bound together by a disulphide bond and it is
also bound to another 3 chains group (in total 6 chains). It has 2 domains – D-domains – and one central
domain – the E-domain. When thrombin acts on fibrinogen, it removes the two tails at the end terminal and
the fibrinogen becomes insoluble and active (fibrin)  the fibrinogen becomes a monomer of fibrin after
the cutting of these two terminal tails, that are released (FIBRINOPEPTIDES).
 By measuring fibrinopeptides A and B we can quantify the amount of fibrinogen converted into fibrin.
 This analysis describes the intensity of coagulation.

- D-dimer quantification
Plasmin, able to digest the fibrin clots, can be active also on fibrinogen. The activity of plasmin favors the
release of D-dimer from the stabilized clot. D-dimer only results from the digestion of stabilized fibrin.
 Normally, the D-dimer is not present neither in the original fibrinogen molecule or in the degradation
products or in soluble fibrin, so it’s a specific product of degradation operated by plasmin on the fibrin
stabilized by factor XIII.

- Quantification of FDP (fibrin/fibrinogen degradation products)

These molecules derive from the action of plasmin of fibrinogen, on fibrin monomer and on unstable
fibrin polymer, without distinction. So we don’t know exactly from where FDPs come from.

N.B. Both the analysis of D-dimer and FDP are useful to describe the intensity of coagulation, but the D-
dimer describes that there is a clot  thrombotic risk  use of preventive drugs  the efficacy of the
treatment can be evaluated by measuring D-dimer.

Markers for the activation of the coagulation system and fibrinolytic system
- ⬆ fibrinopeptides A and b
- ⬆ D-dimer
- ⬆ FDPs
- ⬇ antithrombin III

Reduced level of coagulation and platelet factors:

- Hypofibrinogenemia
- ⬇ in coagulation factors (V and VIII)
- Prolonged PT and aPTT
- Thrombocytopenia