Normal Haemostasis

Presented by: Reem Eshra

Supervised by: Dr. Fatma Afdal
The normal haemostatic response to vascular damage depends on a
closely linked interaction between the blood vessel wall, circulating
platelets and blood coagulation factors
 Platelets

Production Structure

Antigens Function
Platelet production
Thrombopoietin (TPO) is the major regulator of platelet formation and
95% is produced by the liver. As platelets age they lose surface sialic
acid. This exposes galactose residues that attach to the Ashwell–Morell
receptor in the liver. This attachment signals for production of new
TPO. TPO increases the number and rate of maturation of
megakaryocytes via c‐MPL receptor. Platelet levels start to rise 6 days
after the start of therapy. Although TPO itself is not available for clinical
use, thrombomimetic agents which bind to c‐MPL are now used
clinically to increase the platelet count.
Platelet structure
Platelets are extremely small and discoid, 3.0 × 0.5 μm in diameter. The
glycoproteins of the surface coat are particularly important in the
platelet reactions of adhesion and aggregation, which are the initial
events leading to platelet plug formation during haemostasis. Adhesion
to collagen is facilitated by glycoprotein Ia (GPIa). Glycoproteins Ib
(defective in Bernard–Soulier syndrome) and IIb/IIIa (also called αIIb
and β3, defective in Glanzmann’s thrombasthenia) are important in the
attachment of platelets to von Willebrand factor (VWF) and hence to
vascular subendothelium. The binding site for IIb/IIIa is also the
receptor for fibrinogen which, like VWF, is important in platelet–
platelet aggregation.
The platelet contains three types of storage granules: dense, α and
lysosomes (Fig. 24.5). The more frequent specific α granules contain
clotting factors, VWF, platelet‐derived growth factor (PDGF) and other
proteins. Dense granules are less common and contain adenosine
diphosphate (ADP), adenosine triphosphate (ATP), serotonin and
calcium. Lysosomes contain hydrolytic enzymes. Platelets are also rich
in signalling and cytoskeletal proteins, which support the rapid switch
from quiescence to activation that follows vessel damage.
Platelet antigens
Several platelet surface proteins have been found to be important
antigens in platelet‐specific autoimmunity and they have been termed
human platelet antigens (HPA). In most cases, two different alleles
exist, termed a or b alleles. Platelets also express ABO and human
leucocyte antigen (HLA) class I but not class II antigens.
Platelet function

The main function of platelets is the formation of mechanical plugs

during the haemostatic response to vascular injury. In the absence of
platelets, spontaneous leakage of blood through small vessels may
occur. There are three major platelet functions:

Adhesion Aggregation Amplification

 Platelet release
Platelet
vWF reaction &
aggregation
amplification

Platelet Natural
procoagulant Growth factor inhibitors of
activity platelet function
vWF  involved in shear-dependent platelet adhesion to the vessel
wall and to other platelets (aggregation)
 carries factor VIII

Plasma VWF is almost entirely derived from endothelial cells, with

two distinct pathways of secretion. The majority is continuously
secreted and a minority is stored in Weibel– Palade bodies. The
stored VWF can raise the plasma levels when released under the
influence of several secretagogues, such as stress, exercise,
adrenaline and infusion of desmopressin (1‐diamino‐8‐D‐arginine
vasopressin; DDAVP). The VWF released from Weibel–Palade bodies
is in the form of large and ultra‐large multimers, the most adhesive
and reactive form of VWF. They are in turn cleaved in plasma to
smaller multimers and monomeric VWF by the specific plasma
metalloprotease, ADAMTS13.
Platelet release reaction & amplification
Primary activation by various agonists induces intracellular signalling, leading
to the release of α granule contents. These have an important role in platelet
aggregate formation and stabilization and, in addition, the ADP released
from dense granules has a major positive feedback role in promoting platelet
activation. Thromboxane A2 (TXA2) is important in secondary amplification
of platelet activation to form a stable platelet aggregate. It is formed de novo
upon activation of cytosolic phospholipase A2 (PLA2) (Fig. 24.7). TXA2 lowers
platelet cyclic adenosine monophosphate (cAMP) levels and initiates the
release reaction (Fig. 24.7). TXA2 not only potentiates platelet aggregation,
but also has powerful vasoconstrictive activity. The release reaction is
inhibited by substances that increase the level of platelet cAMP. One such
substance is prostacyclin (PGI2), which is synthesized by vascular endothelial
cells. It is a potent inhibitor of platelet aggregation and prevents their
deposition on normal vascular endothelium.
Platelet procoagulant activity
After platelet aggregation and release, the exposed membrane
phospholipid (platelet factor 3) is available for two reactions in the
coagulation cascade. Both phospholipid‐mediated reactions are
calcium‐ion dependent. The first (tenase) involves factors IXa, VIIIa and
X in the formation of factor Xa. The second (prothrombinase) results in
the formation of thrombin from the interaction of factors Xa, Va and
prothrombin (II). The phospholipid surface forms an ideal template for
the crucial concentration and orientation of these proteins.

Growth factor
PDGF found in the α granules of platelets stimulates vascular smooth
muscle cells to multiply and this may hasten vascular healing following
injury.
Natural inhibitors of platelet function
Nitric oxide (NO) is constitutively released from endothelial cells (Fig.
24.9) and also from macrophages and platelets. It inhibits platelet
activation and promotes vasodilatation. Prostacyclin synthesized by
endothelial cells also inhibits platelet function (Fig. 24.9) and causes
vasodilatation by raising cyclic guanosine monophosphate (GMP)
levels. An ectonucleotidase (CD39) acts as an ADPase and helps prevent
platelet aggregation in the intact vessel wall
 Blood Coagulation
Coagulation is initiated after vascular injury by the interaction of the
membrane bound tissue factor (TF), exposed and activated by vascular
injury, with plasma factor VII.

The factor Xa, in the absence of its cofactor, forms small amounts of
thrombin from prothrombin. This is insufficient to initiate significant
fibrin polymerization. Amplification is needed.
The initiation pathway or extrinsic Xase is rapidly inactivated by tissue
factor pathway inhibitor (TFPI). Thrombin generation is now dependent
on the traditional intrinsic pathway. In this amplification phase the
intrinsic Xase, formed by IXa and VIIIa on phospholipid surface in the
presence of Ca2+, activates sufficient Xa, which then, in combination
with Va, PL and Ca2+, forms the prothrombinase complex and results in
the explosive generation of thrombin which acts on fibrinogen to form
the fibrin clot.
Hemostasis:
BV Injury
Tissue
Neural Factor

Blood Vessel Platelet Coagulation

Constriction Activation Activation
Primary hemostatic plug

Reduced
Plt-Fusion Thromibn,
Blood flow
Fibrin

Stable Hemostatic Plug

Blood vessels
Vasoconstriction
• Narrows the lumen of the vessel to minimize the loss of blood
• Brings the hemostatic components of the blood (platelets and
plasma proteins) into closer proximity to the vessel wall
• Enhances contact activation of platlets
• Von Willebrand factor
• Collagen fibers
• Platlet membrane glycoprotein Ib
• Activated platlets enhance activation of coagulation proteins
Endothelial cells
The endothelial cell has an active role in the maintenance of vascular
integrity. This cell provides the basement membrane that normally separates
collagen, elastin and fibronectin of the subendothelial connective tissue from
the circulating blood (Fig. 24.12). Loss of or damage to the endothelium
results in both haemorrhage and activation of the haemostatic mechanism.
The endothelial cell also has a potent inhibitory influence on the haemostatic
response, largely through the synthesis of prostaglandin, NO, and the
ectonucleotidase CD39, which have vasodilatatory properties and inhibit
platelet aggregation. Synthesis of tissue factor that initiates haemostasis only
occurs in endothelial cells following activation, when its natural inhibitor,
TFPI, is also synthesized. Endothelial synthesis of prostacyclin, VWF, factor
VIII, plasminogen activator, antithrombin and thrombomodulin, the surface
protein responsible for activation of protein C, provides agents that are vital
to both platelet reactions and blood coagulation (Fig. 24.12).
Platelet-vessel wall adhesion
Healthy unperturbed endothelial cells exert a powerful inhibitory
influence on haemostasis by virtue of the factors they release or
express on their surface. Two of these, prostaglandin (PG)I2 (or
prostacyclin) and nitric oxide (NO), also known as endothelium-derived
relaxing factor, have powerful vasodilator activity, acting on smooth-
muscle cells in the vessel wall (basal-directed secretion) and hence
modulating blood flow. Both substances inhibit aggregation of platelets
and leucocytes (luminal-directed secretion) by raising intra platelet
levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine
monophosphate (cGMP), respectively.
vWF
VWF is a multimeric glycoprotein that plays an important role in
primary haemostasis by promoting platelet adhesion to the
subendothelium at sites of vascular injury under high shear rate
conditions. It is also a carrier of FVIII and this association protects FVIII
from rapid proteolysis. VWF is synthesized by endothelial cells,
megakaryocytes and platelets. In endothelial cells VWF may be
secreted directly into the circulation or stored in Weibel–Palade bodies.
The VWF produced in megakaryocytes and platelets is not secreted, but
stored in α-granules. Release of VWF from these stores occurs
following activation of endothelial cells or platelets. Some therapeutic
products, such as desmopressin, may work by stimulating release of
stored VWF
 7

Fibrinolysis
Plasminogen
Extrinsic: t-PA, urokinase
Activation
Intrinsic: factor XIIa, HMWK, kallikrein
Exogenous: streptokinase

Fibrin, fibrinogen

Plasmin

Fibrin, fibrinogen
degradation products
 FIBRINOLYSIS
FIBRIN POLYMER

D E D D E D
D E D
D E D D E D

D D
E D-DIMER FIBRIN SPLIT PRODUCTS
D D
INVESTIGATION OF HEMOSTASIS

1. Tests of vascular integrity:

Also affected by thrombocytopenia or
thrombocytopathy.
• Bleeding time (N: 6-10 min). Making small wound in
the skin of the forearm , average time that elicit until
bleeding stop is then measured
• Hess’s test ( Capillary resistance test): Area 5 Cm in
diameter marked on antecubital fossa and
• .
sphygmomanometer is inflated to 80 mm Hg for 5
minutes. Normally 3-5 purpuric eruptions appear.
2. Test for platelets:
• Blood count and film: Show number and morphology
of platelets & any blood disorder as leukemia.
Pseudothrombocytopenia may be due to clumping with
EDTA so use heparin
Pseudothrombocytopenia
• Direct test of platelet function:

• Platelet aggregation: By adding of aggregating agents as ADP, collagen,

thrombin, adrenaline and antibiotic Ristocetin.
• Platelet adhesion: (to glass surface): Decrease in Von willebrand disease,
uremia and thrombocytopenia & increase in arterial and venous disease and
after surgery.
• Platelet antibodies: Its presence indicates autoimmune thrombocytopenia
 3. Tests for blood coagulation

(1)Prothrombin time (PT) (N: 16-18 sec).

It is prolonged with abnormality of factor VII, X, V or
prothrombin and fibrinogen (extrinsic system). It is used
for control of anticoagulant therapy.
(2) Activated partial thromboplastin time (APTT) (N: 30-50
sec).
It is prolonged with deficiencies or inhibitors of I, II, V,
VIII, IX, X, XI, XII. (Intrinsic and common pathway).
(3) Thrombin time (TT) (N: 10-20 sec) : It is prolonged with fibrinogen deficiency and
dysfibrinogenemia or inhibitors as heparin or FDPs.
(4) Plasma fibrinogen level (N: 150-400 mg%)
• Increase in pregnancy & postoperative.
• Decrease in DIC & congenital hypofibrinogenemia.
(5) Coagulation factor assay: Value below 40% is abnormal.
(6) Whole blood clotting time (N: 5-10 min): Normal test excludes cross deficiency in the
coagulation time but neglect minor changes .
(7) Fibrinogen/fibrin degradation products (FDPs) (N:  10 mcg/ml): It is increase in all cases of
DIC and in thromboembolic disease . Very high level above 600 mg/ml occur in severe DIC.
 NORMAL HAEMOSTASIS:

INVESTIGATION OF HEMOSTASIS