Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2617_Библиотеки_им_академика_М_И_Перельмана
.pdf
216
https://t.me/medicina_free
G. Gessoni
Fig. 16.10 Identication of allo-antibodies using an extended identication panel with and without enzyme treatment. This is the same sample already screened, as shown in Fig.16.9. For identication we tested
the sample against a panel of polyantigenic group O red blood cells not
treated with enzymes, the test was performed in Liss-Combs. Some
negative test cells 6, 11 and 12 were observed; such as some positive
tests with different score: ++++ for columns 4, 5, and 13; and +++− for
1, 2, 3, 7, 8, 10, 14, and 15. after this series of tests, based on the results
obtained we hypothesized the presence of an antibody mixture to solve
which we decided to use a different panel and a different reaction
method, as shown in the second series of images. We then tested the
Fig. 16.11 Allo antibody
titration. To carry out the
titration of an antibody, serial
factor two dilutions are
prepared which are tested
against a cell that has been
tested reactive to IAT using
the most appropriate phase.
The dilution of the last
positive column indicates the
reciprocal of the antibody
titer. Results: Titer 1/32
sample against the same panel of polyantigenic group O test red
cells, but enzyme-treated, the test was performed in Liss. As can be
seen, the negativities for cells 6, 11 and 12 remain as well as the positivity ++++ for columns 4,5 and 13. The positivity for cells 1, 2, 3, 7, 8,
10, 14 and 15 disappear; a sign that the antigens to which the antibody
was directed was destroyed by the enzymatic treatment of the red blood
cells. The combined evaluation of the results of the two tests made it
possible to resolve the mixture by identifying an anti-E (specicity that
remains even in the panel treated with enzymes), and an anti-M antibody whose reactivity is eliminated by the enzymatic treatment
Direct Antiglobulin Test (Direct Coombs Test)
The direct antiglobulin test (DAT) is used to detect invivo
sensitization of erythrocytes by γglobulins or complement
fractions. It is rst performed using a polyspecic Coombs
serum. DATpositivity occurs with the presence of at least
500 immunoglobulin molecules attached to the red blood
cells, and can be traced back to one of the following
factors:
• Auto antibodies directed to the patient’s own erythrocyte
antigens
• Allo antibodies present in a recently transfused patient
that react with antigens present on the donor’s red
cells
• Passively acquired antibodies by administration of plasma
or immunoglobulins or, in the case of maternal antibod-
ies, that have crossed the placenta and react with fetal red
blood cells (neonatal hemolytic disease)
16 Immunohematology
https://t.me/medicina_free
217
Fig. 16.12 Identigram. The gure shows the data sheet showing the
antigenic composition of the panel of polyantigenic red blood cells of
group O used for the identication of the antibody mixture shown in
Fig. 16.13 Direct antiglobulin test. Patient shows a marked positivity
(++++) with the polyspecic Coombs serum. The positivity is conrmed for IgG (++++) and for C3b (+++−), the auto-control is negative. The patient therefore presents both IgG and complement on the
• Antibodies directed to drugs that bind to the erythrocyte
membrane (such as penicillin)
• Nonspecic binding (mediated by drugs usually cephalosporins) of immunoglobulins to the erythrocyte
membrane
• Presence of complement fractions on the surface of red
blood cells with a mechanism mediated by drug-induced
immune complexes
• Autoimmune diseases
Figure 16.13 shows two markedly positive samples
for the direct antiglobulin test. The sample above is reactive with polyvalent Coombs serum (AHG.CD), with
monospecific serum for IgG (IgG.IG) and C3d (C3d.IG);
therefore, on the surface of these red cells there are IgG
and complement fractions. The sample below is reactive
with polyvalent Coombs serum and with monospecific
serum for IgG but not for C3d; therefore, on the surface
Fig.16.10. Each row corresponds to the typing of each of the 15 cells
that make up the panel and for each column the positivity or negativity
for each of the antigens is reported
surface of the red cells. The second patient shows marked positivity
(++++) with polyspecic Coombs serum. The positivity is conrmed
for IgG (++++) but both the C3b and the auto-control arenegative. The
patient therefore presents exclusively IgG on the surface of the red cells
of these red cells there are IgG but not complement
fractions.
In case of positive TCD with polyspecic Coombs serum,
the antibody adhered to the red blood cells is then identied
by TCD with monospecic sera and using elution methods.
The direct antiglobulin test with monospecic sera is used to
detect invivo sensitization of erythrocytes by di γ-globulins
or complement fractions. Secondly, monospecic Coombs
sera (anti-IgG, anti-IgA, anti-IgM, anti-C3, anti-C3b, anti C4) can be tested.
The elution methods are used to remove the antibodies
adhered to the red blood cells, and to be able to proceed with
their study using the eluate. Several methods of antibody elution based on physical methods are available: hot elution,
particularly effective for IgM; by freezing and thawing,
usable in cases ofABO related HNFN; or chemical methods:
for example, elution with organic solvents or with acid glycine which is widely indicated in the elution of IgG antibod-
218
https://t.me/medicina_free
G. Gessoni
ies. Once the antibodies have been eluted, it is also possible
to try to identify their specicity.
Applications ofMolecular Biology
toErythrocyte Immunohematology
Traditionally, erythrocyte immunohematology is based on
the study of the phenotype, determined by the search for the
presence on the surface of the red blood cells of antigens
belonging to blood group systems using appropriate antisera.
Direct genotype determination is now routinely applied in
immunohematology.
DNA extraction usually takes place from whole blood
collected in EDTA, using automated methods that can lighten
the workload and at the same time ensure good quality of the
extracted nucleic acids. After the extraction phase, we move
on to the amplication phases of the target with a multiplex
method, which allows the simultaneous and allele-specic
amplication of multiple target sequences.
In the routine of our immunohematology laboratory, commercial gene-chips are usually used for the detection of
amplied products, which allow to study polymorphisms of
different blood group systems at the same time. The Blood
Chip Reference system, in routine use in our laboratory,
allows to determine the genotype of antigens related to ABO,
RH, Kell, Duffy, Dombrock, Colton, Lutheran, Diego Kidd
and MNS blood group systems. The system foresees that the
amplicons, labelled with a uorescent substrate, are detected
by binding with allele-specic probes immobilized on a
slide. The slide is then scanned by a laser, and for each probe
the positivity for the single allele is assessed, based on the
bound uorescence, using specic software.
Figure 16.14 shows genotyping in immunohematology;
the Blood Chip Reference system adopted in our
Laboratory.
Another widespread system is the one called HEA
BeadChip, capable of determining the genotype of antigens
related to the blood group systems ABO, RHD, RHCE, Kell,
Duffy, Dombrock, Lewis, Colton, Scianna, Lutheran, Diego,
Kidd, and MNS.In this system, the amplied products are
denatured and linked, through the use of specic allele
probes, to color-coded microspheres. Upon successful bonding, the probe elongates and produces a uorescent signal
which is detected by an image capture system using a uorescence microscope. The intensity of the signal and the
probe related to it allow to trace the genotype.
At present, genotyping methods nd routine applications
in the extended typing of donors for the establishment of
“banks of rare groups”, which can only be computerized or
provide for the storage of frozen erythrocyte concentrates.
They are widely used in the study of transfusion-dependent
subjects (i.e., patients with hemoglobinopathies), in which
the determinable phenotype no longer corresponds to the
genotype, as well as in the study of rare groups or carriers of
weak antigenic variants. The applications in the eld of prevention of MEN appear to be very interesting, with the possibility of carrying out foetal genotyping both from foetal
cell material obtained with an invasive method and from free
DNA in maternal plasma.
Fig. 16.14 Genotyping in immune hematology; the Blood Chip Reference system adopted in our laboratory
16 Immunohematology
https://t.me/medicina_free
219
Recommended Readings
Anstee D (2009) Red cell genotyping and future pretransfusion testing.
Blood 114:248–252
Anstee D (2011) The functional importance of blood group-active mol-
ecules in human red blood cells. Vox Sang 100:140–149
Blaney K, Howard P (2013) Basic and applied concepts of blood bank-
ing and transfusion practices, 3rd edn. Elsevier
Brown P (1994) Antibody identication. In: Rudman S (ed) Textbook
of blood banking and transfusion medicine. Saunders & co,
Philadelphia
Brown P, Resolving ABO (1994) Typing discrepancies and other typing
problems. In: Rudman S (ed) Textbook of blood banking and transfusion medicine. Saunders & co Philadelphia
Cooling L (2016) Carbohydrate blood groups. In: Rossi’s principles of
transfusione medicine, 5th edn. Wiley Blackwell
Daniels G (2013) Human blood groups, 3rd edn. Wiley-Blackwell,
Oxford
Fung MK, Grossman BJ, Hillyer CD, Westhoff CM (eds) (2014)
Technical manual, 18th edn. AABB, Bethesda
Harmening D (2012) Modern blood banking and transfusion medicine,
6th edn. FA Davis edition, Philadelphia
Hiller S, Ness A, Robak. (2007) Blood banking and transfusion medi-
cine basic principles & practice, 2nd edn. Churchill and Livingstone
USA
Issitt PD, Anstee DJ (1998) Applied blood group serology. Montgomery
Scientic Publications, Miami
Klein HG, Anstee DJ (2005) Mollison’s blood transfusion in clinical
medicine, 11th edn. Blackwell, Malden
McBean R, Hylard C, Flower R (2014) Approaches to determination
of a full prole of blood group genotypes: single nucleotide variant
mapping and massively parallele sequencing. Comp Struct Biotech
J 11:147–151
McCullough J (1998) Transfusion medicine. Mc Grow Hill, NewYork
Rao G, Eastlund T, Jaganathan L (2007) Handbook of blood banking
and transfusion medicine. Japee Medical Edition, New Delhi
Reali G (2009) Immunoematologia e Trasfusione. In: Trattato Italiano
di Medicina di Laboratorio Volume VI.Seconda Edizione Piccin
Reid ME, Mohandas N (2004) Red blood cell blood group antigens:
structure and function. Semin Hematol 41:93–117
Reid ME, Lomas-Francis C, Olsson ML (2012) The blood group anti-
gen factsbook. Academic Press, London
Schenkel-Brunner H (2000) Human blood groups: chemical and bio-
chemical basis of antigen specicity, 2nd edn. Spinger-Verlang,
Vienna
Sorry J (2016) Other protein blood groups. In: Rossi’s principles of
transfusione medicine, 5° edizione edn. Wiley Blackwell
Storry JR, Castilho L, Daniels G et al (2014) International Society
of Blood Transfusion Working Party on red cell immunogenetics
and blood group terminology: Cancun report (2012). Vox Sang
107:90–96
Telen M (2000) Red cell antigen. In: Anderson K, Mess P (eds)
Scientic basis of transfusion medicine, 2nd edn. WB Saunders co,
Philadelphia
Turgeon M (1995) Fundamentals of innumohematology, 2nd edn.
William & Wilkins, Baltimore
Westhoff C (2016) Red cell immunology and compatibility testing. In:
Rossi’s principles of transfusione medicine, 5° edizione edn. Wiley
Blackwell
Westhoff C, Siegel D (2016) Rh and LW blood groups antigens. In:
Rossi’s principles of transfusion medicine, 5° edizione edn. Wiley
Blackwell
Wilkinson S (1994a) The ABO anf H group systems. In: Rudman S
(ed) Textbook of blood banking and trasfusion medicine. Saunders
& co Philadelphia
Wilkinson S (1994b) Other blood group. In: Rudman S (ed) Textbook of
blood banking and trasfusion medicine. Saunders & co Philadelphia
Wilkinson S (1994c) The Lewis, secretor, and soluble ABH antigents.
In: Rudman S (ed) Textbook of blood banking and trasfusion medicine. Saunders & co Philadelphia
Wilkinson S (1994d) The Rh blood group system. In: Rudman S (ed)
Textbook of blood banking and trasfusion medicine. Saunders & co
Philadelphia
Hemostasis
Procoagulant
Procoagulant
Anticoagulant
Anticoagulant
Hemorrhage Thrombosis
https://t.me/medicina_free
GiuseppeLippi, GianLucaSalvagno,
andMassimoFranchini
17
Introduction
Hemostasis is a complex balance in which anticoagulant
forces contribute to maintaining the fluidity of the blood
within intact vessels and procoagulant forces act instead
to avoid an excessive outflow of blood when the vessels
lose their integrity (Fig.17.1). Hemostasis can be divided
into primary, in which platelets and the vascular system
play a key role, and secondary, in which coagulation factors participate. The subsequent fibrinolytic process has
the function of bringing the vessel back to patency when
the lesion has been repaired. This chapter will describe
the main pathophysiological mechanisms of hemostasis
and the related laboratory tests useful in the diagnosis
and therapeutic monitoring of the main coagulation
disorders.
G. Lippi (*)
Section of Clinical Biochemistry, University Hospital of Verona,
Verona, Italy
e-mail: giuseppe.lippi@univr.it
G. L. Salvagno
Section of Clinical Biochemistry, Department of Neurological,
Biomedical and Movement Sciences, University Hospital of
Verona, Verona, Italy
M. Franchini
Department of Hematology and Transfusion Medicine, Carlo Poma
Hospital, Mantua, Italy
forces
Hemostatic
balance
forces
Fig. 17.1 Hemostatic balance. (Copyright EDISES 2021. Reproduced
with permission)
forces
forces
Vascular System
The vascular system in mammals, including humans, is
divided into four types, including arterial vessels, venous
vessels, capillary vessel systems, and lymphatic systems,
which are described below.
Arterial vessels (the arteries): Their function is to transport blood from the heart to the peripheral organs to carry
oxygen, nutrients, and other molecules. Their function is
essential to ensure the survival of the organs. They are, therefore, characterized by a very elastic wall (to allow the propagation of the sphygmic wave) and very high internal pressure
(about 100–140 mmHg during systole and about
60–90mmHg during diastole) to ensure efcient perfusion.
Venous vessels (veins): They have the function of conveying blood from the peripheral organs to the heart (“venous
return”). They have much thinner walls than the arteries, and
the ow of blood inside them is guaranteed by a residual
arterial pressure (5–10mmHg) and by the contractile action
of the muscles that often surround them.
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_17
221
222
https://t.me/medicina_free
G. Lippi et al.
Capillary vessel system (capillary system): It represents
the junction point between arteries and veins. Capillaries are
essentially characterized by a small caliber (5–10μm, sufcient for transit), very thin walls, low pressure (35–40mmHg
at the arterial end and 15−20mmHg at the venous end), and
low speed of blood ow (about 1mm/second).
Lymphatic system (lymphatic vessels): It is a system of
drainage vessels whose function is to carry the lymph from
the interstitial space to the circulatory stream. It does not
contain blood or even components of the hemostatic system
but instead contains proteins and lipids.
Hemostatic Balance
The main hemostatic events, at least those of clinical relevance, occur in most cases in arterial and venous vessels. An
imbalance in the hemostatic system can lead to two types of
events: thrombosis, dened as a procoagulant tendency of
the blood within an intact vascular system, and hemorrhage,
dened as an inefcient coagulative capacity within a vascular system that has lost integrity.
A minimum degree of thrombosis can physiologically
occur within intact vessels (e.g., as a result of a slight
trauma), such, however, as never to exceed the effect of anticoagulant forces to prevent excessive amplication and
transformation into a process of pathological thrombosis.
Briey, in a normal subject, thrombosis is effectively
stemmed. Pathological thrombosis occurs, therefore, only
when the procoagulant forces exceed the anticoagulant ones
in circumstances where this is not necessary (intact vessel).
It can be due to an excess of procoagulant forces (excess of
prothrombotic factors) or a defect of anticoagulant forces
(decit of antithrombotic forces).
Depending on the extent of vascular damage, a variable
degree of hemorrhage occurs physiologically after the rupture of a blood vessel (e.g., because of small cuts or wounds)
and can involve a vein, an artery, or even the capillary system. Nevertheless, hemorrhage is usually counteracted by a
series of mechanisms that will be dened in detail below.
Briey, in the presence of an efcient hemostatic system and
compatible with the size of the damage, an abnormal (excessive or pathological) hemorrhage occurs when the anticoagulant forces overwhelm the procoagulant ones after the rupture
of an intact vessel, and this is usually due to a defect of procoagulant forces (decit of prothrombotic factors) or to an
excess of anticoagulant forces (excess of antithrombotic factors). It is also easily comprehensible that the hemorrhagic
phenomenon assumes different characteristics in relation to
the type of vessel involved. In the veins, the pressure of the
blood inside the vessels is such that the hemorrhage resulting
from lesions of large caliber veins can be effectively counteracted by the combined action of the hemostatic system and
external therapeutic interventions, such as compression or
ligation of the vessel (obviously, potential “internal” hemorrhages that are not recognized are exceptions). On the contrary, the pressure and the ow rate in the arteries (especially
in those of large caliber) are such that in the presence of a
lesion in a femoral and/or humeral artery, for example, the
hemostatic system cannot stop the bleeding and, at the same
time, even external compression or “ligation” maneuvers can
sometimes be ineffective, leading the patient to death for
bleeding. Nevertheless, if the hemostatic balance is seriously
altered in favor of an anticoagulant status, the loss of integrity of small arterial vessels and/or veins can lead to severe
hemorrhages that cannot be found in subjects with normal
hemostatic function.
Hemostasis
Hemostasis is conventionally divided into three phases: primary (also known as the vessel-platelet phase), secondary
(also known as the coagulation cascade) (Table 17.1), and
brinolysis. The rst two phases contribute to the formation of
a stable hemostatic plug (also known as thrombus) to prevent
the leakage of blood from the damaged vessels, while brinolysis determines the lysis of the hemostatic plug when it is no
longer necessary (restitutio adintegrum of the vascular wall).
Primary Hemostasis
Primary hemostasis is dened as the process that determines
the formation of a rst bulwark against hemorrhage to prevent the “acute” leakage of blood from the vessels. The main
effectors of this phase are the vessels (endothelium and muscle cells) and platelets.
Vessel Participation
The participation of the vessels in the hemostatic process is
often underestimated, but it is far from secondary. The wall
of the blood vessels is composed of a concentric series of
layers, including the tunica intima (composed of endothelium and subendothelial layers), the tunica media (with a
prevalence of elastic and muscular bers in the arteries and
brous tissue in the veins), and the tunica adventitia (composed mainly of dense connective tissue). Injury to the vessel
reexively causes contraction of the elastic and muscular
bers of the middle layer, a process aimed at reducing the
caliber of the vessel and its output. This phenomenon, also
known as vasospasm, is mediated by thromboxane A2
(TXA2) and can also be activated secondary to neurogenic
stimuli such as pain. The reduced blood ow within the contracting vessel is therefore reected in a reduction in the
amount of blood leaving the vessel following its rupture.
17 Hemostasis
https://t.me/medicina_free
223
Table 17.1
Number First name Origin Function
FI Fibrinogen Liver Precursor of brin; stabilizes the clot
FII Prothrombin Liver Precursor of thrombin; activates brinogen, FV, FVIII, FXI, and
FIII Tissue factor (TF); tissue
FIV (Ca) Calcium
PV Proaccelerin; labile factor
FVII Proconvertine; stable factor Liver It binds the TF to form the extrinsicase complex which activates
FVIII Anti-hemophilic A factor Endothelium FIXa cofactor in the tenase complex that activates FX
FIX Anti-hemophilic B factor;
FX Stuart-Prower factor Liver It binds to the FV to form the prothrombinase complex; activates
FXI Anti-hemophilic C factor;
FXII Hageman factor Liver Activates precallicrein and FXI; activator of the intrinsic
FXIII Fibrin stabilizing factor Liver Activated by thrombin; catalyzes the formation of covalent
PK Precallicrein; Fletcher factor Endothelium Activate the FXII
HMWK High molecular weight
VWF Von Willebrand factor Endothelium and
Numbering of coagulation factors (F)
thromboplastin
Christmas factor
antecedent plasma
thromboplastin
Kininogen; Fitzgerald factor
FXIII and binds to thrombomodulin to activate protein C
Endothelium Contained in perivascular broblasts and epithelial cells; it binds
Platelets, α granules
Liver It binds to FIX to form the tenase complex; activate FX
Liver Activate FIX
Liver and endothelium Cofactor of the intrinsic pathway and mediator of inammation;
megakaryocytes
to FVII to form the extrinsicase complex which activates the
coagulation cascade
Mediates the binding of the gamma- carboxylic end groups of
the coagulation factors to the phospholipids of the platelet wall
FXa cofactor in the prothrombinase complex that activates
prothrombin
the coagulation cascade
prothrombin
pathway (mainlyin vitro)
bonds between two adjacent brin monomers, to make the brin
and clot stable
it has no intrinsic catalytic activity
It binds and stabilizes the FVIII; mediates platelet aggregation
Simplifying the concept, the less blood circulates within the
vessel, the less chance it has of escaping from the point
where the damage occurred.
It is easy to understand that vascular contraction is very
efcient in the arterial district (since in the arteries the middle layer is much thicker), while its efciency is obviously
much lower in the venous district, in which the middle layer
consists mostly of brous tissue. Nevertheless, this difference has a valid pathophysiological explanation: since the
pressure in the arteries is much higher than in the veins, a
much more effective vascular contraction is required in the
arteries in order to prevent a considerable leakage of blood
from the vessel. The functions of vasoconstriction can be
summarized in three decisive aspects:
• Reduction of the ow rate of the vessel, thus limiting the
amount of blood that can ow out of it
• Promotion of platelet margination at the site of endothe-
lial damage and near the injured area
• Promotion of the accumulation, at the site of endothelial
damage, of coagulation factors to enhance the activation
and propagation of secondary hemostasis
The vascular endothelium takes part in the hemostatic
processes with two prevalent activities: the liberation of
tissue factor (Tissue Factor, TF), following the rupture of
the cytoplasmic membrane, and the active production of
Von Willebrand factor (VWF), contained in the so-called
Weibel–Palade organelles, which will be discussed later
in relation to secondary hemostasis. Accessory roles
played by the endothelium are those related to the inhibition of hemostasis, especially when it is not necessary
(intact vessel). In this context, the anti-hemostatic effect
of the endothelium is mediated by a series of activities,
including:
• Formation of an anatomical barrier to the subendothelium
• Coating with heparinoid substances, which help inhibit
the coagulation cascade.
• Coating with thrombomodulin, which modulates the acti-
vation of the coagulation protein C and S complex
• Production of antiplatelet substances, such as prostacy-
clin (PGI-2) and nitric oxide (NO)
• Production of tissue plasminogen activator (t-PA), whose
role is to activate brinolysis
224
Integrin α2β1
https://t.me/medicina_free
Platelets
Platelets (also known as thrombocytes, from the Greek
θρόμβος, thròmbos, “lump” and κύτος, cytos, “cell”) are cor-
puscular elements of the blood without a nucleus. Platelets
derive essentially from the lysis of the cytoplasm of bone
marrow cells called megakaryocytes (each megakaryocyte
produces about 4000 platelets), are round or oval in shape,
with a diameter of about 1–3μm, and circulate in the blood
in a variable number, generally between 150–400× 109/L,
with a half-life of about 5–9days and elimination mainly by
splenic catabolism. Even if they are “terminal” elements, as
they have no nucleus, these cellular fragments contain inside
them three types of granulations, which represent the prerequisite for their functioning. Briey, the platelet granules are
subdivided into α, δ, and γ granules, as described below.
Subendothelial matrix
gp IIb/IIIa
gp IIb/IIIa
ADP
gp VI
G. Lippi et al.
gp Ib-IX-VFibrinogen
VWF
gp Ib-IX-V
TXA2
• Granules α (alpha): Slightly opaque, very numerous, and
contain mainly brinogen, VWF, factors V and XIII,
bronectin, platelet factor 4 (FP4), β-thromboglobulin
(β-TG), thrombospondin, and various growth factors,
including platelet-derived growth factor (PDGF) and
transforming growth factor (TGF)-β
• δ (delta) granules or “dense” granules: Contain mainly
histamine, serotonin, calcium, ADP, and ATP
• γ (gamma) or lysosomal granules: Contain mainly lysosomal hydrolases and peroxisomes
As previously described, platelets play an essential role in
physiological hemostasis, representing the main effectors of
primary hemostasis. Briey, the role of these small corpuscular elements is to form a rst “plug” (called “platelet plug,”
formed by the “weak” association of platelets) or bulwark to
the leakage of blood following the loss of integrity of the
vessel.
To briey summarize the cascade of events, following
vascular damage, the platelets are exposed to the subendothelium (collagen, proteoglycans, bronectin, and other glycoproteins), and this causes their activation. The platelet
response is implemented through biochemical, structural,
and morphological alterations. The response to a stimulus is
due to the coordinated intervention of the membrane, granules, and cytoskeleton and can be divided into several phases,
which may overlap (Fig.17.2).
Phase I: Adhesion (to theSubendothelium)
The adhesion of platelets to the subendothelium occurs
mainly by binding to the subendothelial collagen, which is
exposed following the loss of integrity of the vascular wall
(rupture of the endothelial layer). The binding between
platelets and collagen is mediated not only by a receptor
located on the platelet membrane, the glycoprotein Ia/IIa (gp
Ia/IIa), but also by the bridging function exerted by VWF,
which binds with a part of the molecule to the collagen and
Fig. 17.2 Primary hemostasis. (Copyright EDISES 2021. Reproduced
with permission)
with another part to a receptor located on the platelet membrane, the glycoprotein Ib (gp Ib). The binding of the platelet
to the subendothelium is also mediated by other platelet
membrane receptors, such as the glycoprotein Ic/IIa (gp Ic/
IIa), which binds laminin and bronectin, and, most likely,
by the glycoprotein IV (gp IV), which binds to collagen by
means of a bridge-bond with thrombospondin. The activation of these platelet receptors is an essential prerequisite for
the subsequent phases of the process, which consist of the
change in the structure of the platelet (shape change) and its
contextual activation. The binding of these rst platelets to
collagen (through the Ia/IIa receptor) and to VWF (which in
turn acts as a bridge for further binding to collagen) causes a
deformation of the three-dimensional structure of the platelets; this change facilitates further aggregation of the platelets among themselves and above all stimulates the so-called
release reaction.
Phase II: Shape Change
The change in platelet shape (from roughly circular and/or
ovaloid elements to a very irregular, almost starry shape,
characterized by pseudopod generation) is sustained by the
contraction of the platelet cytoskeleton and is a propaedeutic
event to the granule release reaction. Of particular interest is
the phenomenon known as the “ip-op” of phosphatidylserines, in which FP4 is exposed on the platelet surface and
represents the physiological binding site of coagulation factors during their activation process (described in detail
below).
Phase III: Activation andSecretion ofGranules
(Release)
The activation of the platelet and the consequent release of
internal granules (through the open canalicular system by
17 Hemostasis
https://t.me/medicina_free
225
direct exocytosis) are aimed at releasing the numerous mediators contained within the three different types of granules
and at enhancing primary hemostasis. This process can be
summarized with the term autocrine activation, a mechanism
according to which a cellular element (the platelet) produces
mediators (mainly ADP and serotonin) that have the function
of activating other identical cellular elements (other platelets), thus amplifying the subsequent phenomenon, platelet
aggregation. It is also important to remember that the release
of TXA2 by the platelets contributes to further stimulating
vasoconstriction and thus limiting the caliber of the vessel
and the outow of blood.
Phase IV: Aggregation (Between Them)
Following the contraction of the cytoskeleton (shape change),
activation, and secretion of granules, platelets express on
their membrane surface the glycoprotein IIb/IIIa (gp IIb/
IIIa), which specically binds brinogen. Since two contiguous activated platelets express gp IIb/IIIa at the same time, it
is not difcult to understand how they can form bridges
between them (gp IIb/IIIa-brinogen-gp IIb/IIIa), which,
therefore, represents the prerequisite for the phenomenon
known as platelet aggregation. Thrombospondin also seems
to play a role in this process, increasing the size of the platelet aggregate by forming a bridge (cross-link) between platelets and brinogen or by positioning itself directly as a bridge
between gp IIb and IIIa of two adjacent platelets. Interestingly,
platelet aggregation occurs in two phases. Primary aggregation is promoted by a modest concentration of agonists
(ADP, collagen, thrombin, and platelet-activating factor) and
is essentially reversible, whereas secondary aggregation is
determined by higher concentrations of agonists (ADP and
TXA2).
The combination of all the phenomena described above,
in the absence of quantitative or qualitative platelet abnormalities, effectively contributes to limiting the leakage of
blood from the damaged vessel within a few minutes. The
platelet plugs thus formed, however, do not have stability
requirements to allow a permanent interruption of blood
leakage, especially in vessels with higher blood ow and
high pressure. The subsequent stabilization of the hemostatic
plug will be guaranteed by the intervention of secondary
hemostasis, described in detail in the next paragraph.
Platelet Disorders
Although a detailed description of platelet diseases is beyond
the scope of this chapter, it may be useful to recall that, like
many other human diseases, they are essentially subdivided
into quantitative (decrease in the number of platelets, also
known as thrombocytopenia) or qualitative (altered platelet
function) diseases, which are in turn subdivided into congenital (or hereditary) and acquired diseases. The principal
platelet pathologies are described in Table17.2. It should be
Table 17.2 Main platelet pathologies
Quantitative (Thrombocytopenias)
Congenital
Wiskott–Aldrich syndrome
May–Hegglin syndrome
Bernard–Soulier syndrome
Acquired
Reduced/impaired bone marrow production of megakaryocytes
Bone marrow aplasia (due to cytotoxic drugs, radiation therapy,
viruses, or other microorganisms)
Medullary neoplasms
Medullary neoplastic inltration
Myelodysplastic syndrome
Deciencies
Vitamin B12 deciency
Folic acid deciency
Increased destruction in circulation
Thrombotic thrombocytopenic purpura (Moschowitz syndrome)
Idiopathic thrombocytopenic purpura (Werlhof disease)
Hemolytic-uremic syndrome
Thrombocytopenic purpura associated with autoimmune
diseases (e.g., autoimmune hemolytic anemia or Fisher–Evans
syndrome, collagenopathies, and sarcoidosis)
Thrombocytopenic purpura associated with lymphoproliferative
diseases (e.g., chronic lymphocytic leukemia, Hodgkin’s and
non-Hodgkin’s lymphomas, and paraproteinemia)
Thrombocytopenic purpura from drugs (e.g. heparin,
isopropylcarbamide, chlorothiazide, sulfonamides, para-
aminosalicylic acid (PAS), quinine, and quinidine)
Thrombocytopenic purpura from isoantibodies (maternal–fetal
immunization and transfusions)
Anti-phospholipid antibody syndrome
Eclampsia
Disseminated intravascular coagulation
Vascular prostheses
Extra-corporeal circulation
Kidnapping
Splenomegaly (hypersplenism)
Giant hemangiomas (e.g., Kasabach-–Merrit syndrome)
Pseudothrombocytopenia (spurious thrombocytopenia due to
sampling issues or induced by ethylenediaminetetraacetic acid,
cryoglobulins, platelet satellites, etc.)
Qualitative (platelet disorders)
Congenital
Bernard–Soulier syndrome (platelet adhesion defect)
Glanzmann’s thrombasthenia (platelet aggregation defect)
Storage pool disease (defect in the release of granules)
Aspirin-like defects (defects in cyclo-oxygenase or thromboxane-
synthetase enzymes)
Gray platelet syndrome (lack of α granules).
Acquired
Excess of brin breakdown products
Monoclonal gammopathies
Type I glycogenosis
Drugs (antiplatelet agents, aspirin, dipyridamole, indomethacin,
phenylbutazone, chlorpromazine, dextran, and ethyl alcohol)
Uremic syndrome
Myeloproliferative syndrome
noted that thrombocytopenia with platelet counts greater
than 50×10
9
/L is usually asymptomatic, 30 to 50×109/L is
usually accompanied by mild bleeding symptoms, and 10 to
30×109/L is associated with moderate bleeding symptoms,
226
platelet
https://t.me/medicina_free
G. Lippi et al.
whereas a decrease in platelet counts to values <10×109/L
frequently results in spontaneous mucosal skin bleeding,
generalized purpura, and the risk of severe urinary, gastrointestinal, and central nervous system bleeding.
Secondary Hemostasis
The purpose of secondary hemostasis is to generate a brin
network to “cement” the primary platelet plug.
Secondary hemostasis, also known as coagulation, consists of a complex series of events involving a large number
of molecules (Table17.1). The traditional model of secondary hemostasis is that of a “cascade,” which describes coagulation as a sequential series of enzymatic or proteolytic
reactions in each of which an inactive precursor of a proteolytic enzyme (termed zymogen) is converted to its active
form (protease), which in turn activates the next zymogen in
the series. Since the individual sequential reactions are catalyzed by an enzyme, and since each enzyme can catalyze the
generation of a large number of other active molecules, it is
easy to understand how this model is characterized by an
enormous amplifying potential, hence the name “cascade.” It
is possible to summarize the main features of the cascade
model in a few essential points.
• Each step in the cascade includes a serine protease and
sometimes a cofactor (to form an enzyme “complex”).
• Enzyme complexes consist of similar components with
similar characteristics with respect to their assembly and
function.
• The formation of the complex results in a considerable
increase (about 100-fold) of the catalytic efciency
towards the substrate.
• To function effectively, individual zymogens and enzyme
complexes require the binding of one of their terminal
carboxyl groups to an appropriate surface consisting of
negatively charged phospholipids (generally those pres-
ent on the membrane of platelets), a binding mediated by
the indispensable presence of calcium ions (Ca2+)
(Fig.17.3). In the absence of the carboxylic group on the
coagulation factors or as a result of the subtraction of cal-
cium ions, the binding between the coagulation factor and
the platelet occurs with much less efciency, overall, also
altering the effectiveness of the coagulation cascade.
During the last two decades, the model of the coagulation
cascade has undergone considerable interpretative revisions,
conditioned by the progressive discovery of new coagulation
factors and new and complex biochemical interactions. The
historical paradigm of coagulation is the one that, for years,
was based on the existence of three different “pathways,”
Coagulation factor
–
COO
+
Ca
+
–
PO
Fig. 17.3 Role of calcium in secondary hemostasis. (Copyright
EDISES 2021. Reproduced with permission)
Activation of
Coagulation
TF + FVIIa
TFPI
PC-PS
FX
Fig. 17.4 Secondary hemostasis. The dotted lines dene the critical
processes of the thrombin burst. AT antithrombin, FB brin, FBG
brinogen, FDP brin degradation products, FII prothrombin, FIIa
thrombin, PC protein C, PG plasminogen, PN plasmin, PS protein S, TF
tissue factor, tPA tissue plasminogen activator, TFPI inhibitor of the
tissue factor pathway. (Copyright EDISES 2021. Reproduced with
permission)
FVa + FXa
FII FIIa
Activation
Inhibition
FBG FDP
FX FXIa
AT
TAFI
FXIII FXIIIa
FB
TAFIa
sol
Thrombin
Burst
PC-PS
FIXa + FVIIIa
FXI
PN PG
FB
st
FIX
tPA
also known as extrinsic, intrinsic, and common. Over the
years, however, it has been demonstrated that this model,
while maintaining stringent validity in the interpretation of
laboratory abnormalities of the coagulation cascade, has no
real feedback invivo. In this case, it has been concluded that
the “parallel” model, according to which extrinsic and intrinsic pathways concur in parallel to the activation of the common pathway, has no real biological correspondence since
the physiological mechanism of activation of coagulation is
represented only by the extrinsic pathway. According to the
most current model, therefore, coagulation can be described
in three sequential phases (Fig.17.4):
Соседние файлы в папке Библиотека им академика М.И. Перельмана