PATHOPHYSIOLOGY OF
PRIMARY AND SECONDARY
HEMOSTASIS. FIBRINOLYSIS.
November 7, 2017
HEMOSTASIS
 The normal physiological response that
prevents significant blood loss following
vascular injury is called haemostasis.6
Familiarity with haemostasis lays the
groundwork for a thorough understanding of
the major disease states associated with
thrombosis, such as venous thromboembolism
(VTE), atherothrombosis (thrombosis triggered
by plaque rupture), and cardioembolic stroke.
HEMOSTASIS
Subendothelial matrix
Platelets
Hemostatic plug
Fibrin
Endothelial cell
RBC
WBC
WBC
CLOT FORMATION
Fibrin
Red Blood Cell
Platelet
ABNORMAL HAEMOSTASIS
 Excessive coagulation leads to the formation
of a thrombus, potentially obstructing blood
flow. This is a common problem, especially in
hospitalised or immobilised patients.
 Venous thromboembolic disease, for example,
is a major problem in the European Union,
where it causes more than one million events
or deaths every year.
Excessive bleeding results when certain
coagulation factors are lacking, as in patients
with haemophilia.
BLOOD VESSEL INJURY
 Blood vessel injury triggers the following sequence:
 The vessel constricts to reduce blood flow
 Circulating platelets adhere to the vessel wall at the site of trauma
 Platelet activation and aggregation, coupled with an intricate
series of enzymatic reactions involving coagulation proteins,
produces fibrin to form a stable haemostatic plug

This finely tuned process serves to maintain the integrity of the
circulatory system.However, the process can go out of balance,
leading to significant morbidity and mortality.
Adhesion
GpIIb/IIIa
PLATELET ACTIVATION PATHWAYS (1)
GpIIb/IIIaGpIIb/IIIa Aggregation
ADP
Adrenaline Platelet
Exposed Collagen
Endothelium
vWF
COLLAGEN
GpIIb/IIIaGpIIb/IIIa AggregationGpIIb/IIIaGpIIb/IIIa Aggregation
AdhesionAdhesion
ADP
Adrenaline
THROMBIN
Platelet Activation Pathways (2)
Platelet Aggregation
Fibrinogen
Fibrinogen Binding Site
Thrombin
Platele
t
Herbert. Exp Opin Invest Drugs 1994;3:449-
455.
THE COAGULATION CASCADE
 Coagulation involves a complex set of
protease reactions involving roughly 30
different proteins.
 The final result of these reactions is to convert
fibrinogen, a soluble protein, to insoluble
strands of fibrin. Together with platelets, the
fibrin strands form a stable blood clot.
Palta S et al., Indian J Anaesth, 58:515-523, 2014
„CELL-BASED MODEL“
 This model identifies membranes of cell presenting tissue
factor )TF) and a surface of platelets as places of activation of
specific coagulation factors.
 The model supposes the model of zhree phases: initiation,
amplificatopn (propagation) and the proper action of
thrombin- thrombus formation.
 Initiation = formation of complex TF-FVIIa which is leading to
avtivation of a small amount of thrombin.
 Propagation = activation of platelets by thrombin and
formation of complex FIXa-FVIIIa with subsequent activation of
factor Xa.
 Thrombus formation = formation of prothrombinase complex
anf of large amount of thrombin which is leading to formation
of thrombus.
INICIATION PHASE OF COAGULATION
Palta S et al., Indian J Anaesth, 58:515-523, 2014
INITIATION PHASE OF COAGULATION
 Coagulation cascade is activated when defect of
vessel wall enables contact of the blood with cells
with TF.
 Platelets membrane bound tissue factor TF activates
FVII to VIIa which is leding to formation of complex TF-
VIIa.
 The complex binding on platelets membranes
activates Factor IX(a) and Factor X(a).
 Factor Xa converts small amount of prothrombin
(Faktor II) on trombin (Factor IIa) which can activate
Factor V on FVa and Factor VIII on FVIIIa.
PROPAGATION PHASE OF COAGULATION
Palta S et al., Indian J Anaesth, 58:515-523, 2014
PROPAGATION OF COAGULATION: CENTRAL
ROLE FOR FACTOR XA
 Factor Xa together with activated Factor V (Va) as
cofactor support coagulation by thrombin formation
(Factor IIa ) from prothrombin (Factor II).
 Factor Xa is primary pount for propagation of the
porcess; one molecule of Factor Xa catalyses
formation of about 1,000 molecules of thrombin.
FINAL STEP: FIBRIN FORMATION
 In the final step, sequence of serin proteinases
reactions which lead to formation of blood
clot, thrombin will convert soluble fibrinogen to
insoluble fibrin.
 Thrombin also activates Factor XIII (stabilizing
fibrin) which can stabilize clot by crosslinking of
fibrin.
 Stabilized fibrin is able to retain cellular
components (red blood cells, platelets or
both).
Fibrinolysis
Palta S et al., Indian J Anaesth, 58:515-523, 2014
Coagulation inhibitors
 Adequate coagulation will develop only on surface of activated cells
(platelets and endothelial cells). Non- activated cells distally of the injury
exprime another inhibitory systems - tissue factor pathway inhibitor (TFPI),
heparansulphate (HS), thrombomodulin (TM) and protein C (PC).
 TFPI prevents thrombin formation on the surface of endothelial cells by
inhibition of FXa an FVIIa formed by cells producing TF (monocytes). By
this, activity of Xa is even in initiation phase strongly controlled.
 Endothelial cells produce HS binding plasma AT and accelerate its
inhibitory activity. As result, inactivation of thrombin and other activated
factors (FXa, FIXa, FXIa, FXIIa) from the space of coagulum formation can
be expected. Heparin is functioning like HS. Formatted thrombin will bind
on TM and the complex thrombin-TM quickly activates PC on activated
PC (APC). APC together with its cofactor protein (PS) irreversibly cleaves
FVa and FVIIIa. This step limits formation of thrombin in propagation
phase. Endothelial cells produce ADPase on their luminal surface which
blocks proaggregation effect of ADP releasing from activated platelets.
NATURAL INHIBITORS OF COAGULATION
 „Tissue factor pathway inhibitor“ –produced by
endothelial cellls. It inhibits complex TF-VIIa.
 Antithrombin (previously AT III) – binds
activated vitamin K dependent coagulation
factors (can be activated by heparin which
increases its binding capacity)
 „Protein Z dependent protease inhibitor/
protein Z (PZI)“ produced by liver. It inhibits FXa
in presence PZ and Ca++.
COAGULATION FACTORS -STATE
 Non activated-after their synthesis in liver
 Posttranslationally modified –vitamin K
dependent coagulation factors = serin
proteázy
 Activated –activated serin proteases,
other activated factors (Va, VIIIa)
ANTICOAGULATION THERAPY
 Vitamin K antagonists (warfarin) block recyclation of oxidased K to
reduced KH2 in liver. Vitamin KH2 is a cofactor of carboxylation of
glutamate residua in N-terminal regions of vitamin K dependent
coagulation factors (serin proteinases). This limits binding of non
carboxylated coagulation using Ca++ on phospholipid surfaces,
especially on surface of activated plateles and endothelial cells.
 Optimal doses of warfarin has great interindividual variability. They are
dependnt on diet, treatment, age, body weight and important genetic
factors [VKORC1 and cytochrom P450 (CYP) 2C9].

TESTS
 Screening tests
 Bleeding time
 Platelet count
 Prothombin time (PT)
 Partial thromboplastin time (PTT)
 Thrombin time (TT)
 More specific tests
SAMPLING
 Venous blood
 Excessive stress and exercise cause changes in blood clotting.
and fibrinolysis.
 Whenever possible, venous samples should be collected
without a pressure cuff (to avoid haemoconcentration,
increase of fibrinolysis, platelet release, and activation of some
clotting factors.
 To minimize the effect of contact activation plastic or
polypropylene, siliconized glass, syringes and containers
should be used.
 Thoroughly mixing the blood with the anticoagulant by
inverting the containers several time.
 The sample should be brought to the laboratory as soon as
possible.
 Labeling the patient sample is very important.
SAMPLING
 Anticoagulant trisodium citrate 3.2 % in a ratio of 1 : 9.
 Time of sample collection is very important factor in the
interpretation of results.
 Centrifugation and preparation of platelets poor plasma -
4000 rpm in a cooling centrifuge.
 P.T & Factor VII  kept at room temperature.
 Other assays  at 4oC.
 Testing should preferably be completed
within 2 hours of the collection.
BLEEDING TIME
 Time taken for bleeding to cease from a
small superficial wound
 Affected by
- Platelet count and function
- Vessel wall
- Normal range Ivy’s method: 2-7 min
PLATELET COUNT
 Normal platelet count = 150-400x109lL
 A part of complete blood picture (CBC)
 Performed by electronic counters or
manually (inherent error)
PROTHROMBIN TIME
 Indicates the overall efficiency of
extrinsic pathway of blood coagulation
(FVII, FII, FV, X)
 Normal range: 10-14 sec
PROTHROMBIN TIME
 Causes of prolonged PT
- Liver disease
- Vit K deficiency (FII, V, VII, IX are Vit k
dependent)
- Deficiency of factors involved in extrinsic
pathway
- DIC
- Oral anticoagulants
PARTIAL THROMBOPLASTIN
 Indicates the overall efficiency of intrinsic
pathway of blood coagulation (FVIII, FIX,
FXI, FXII, FII, FV, X)
 Normal range: 30-40 sec
PARTIAL THROMBOPLASTIN
 Causes of prolonged PTT
- Deficiency of factors involved in intrinsic
pathway (coagulation factors other than
FVII)
- Liver disease
- DIC
- Massive transfusion (labile FV, FVIII)
- Heparin
PT & PTT
 Prolonged PT + normal PTT= extrinsic
pathway defect
 Prolonged PTT + normal PT= intrinsic
pathway defect
 Prolonged PT and PTT= common
pathway defect or combined factor
deficiencies
THROMBOCYTOPENIA
 Platelet count below 150x109/L
 Causes:
 - Congenital
- Acquired
- failure of production
 Increased destruction (ITP)
 - Splenic sequestration (hypersplenism)
IDIOPATHIC THOMBOCYTOPENIC PURPURA
 ITP is immune thrombocytopenia due to
formation of antibodies against platelets and
BM megakaryocytes.
 Clinical picture: spontaneous bleeding purpuric
eruptions.
 BT: prolonged
 Platelet count: thrombocytopenia
 PT,PTT: normal
 BM: increased megakaryocytes with poor platelet
separation
QUALITATIVE PLATELET DEFECT
 Platelet function defect + normal plt
count
 Causes:
- Hereditary (Glanzmann’s disease,
Bernard-Soulier syndrome)
- Acquired (drugs as aspirin, uremia)
QUALITATIVE PLATELET DEFECT
 Clinical picture: spontaneous bleeding
purpuric eruptions.
 BT: prolonged
 Platelet count: normal or slightly decreased
 PT,PTT, TT: normal
 Platelet function: abnormal depending on the
defect (defective aggregation in Glanzmann’s
disease and Bernard-Soulier syndrome)
HEREDITARY THROMBOPHILIA
 Hereditary thrombofilia
 AT deficiency
 Protein C deficiency
 Protein S deficiency
 Factor V Leiden
 Prothrombin polymorphism (G/A 20210 in
3´ area of the gene)
ACQUIRED THROMBOTIC DISORDERS
 Sy of antiphospholipid antibodies
 Increased levels of factors VIII, IX, XI and
fibrinogen
 Fibrinolysis defects
 LMWH
• Bleeding
• Thrombocytopenia
• Hypersensitivity
HEPARIN/LMWH—ADVERSE EFFECTS
 Heparin
 Bleeding
 Thrombocytopenia
 Osteoporosis
 Hypersensitivity
WARFARIN—ADVERSE EFFECTS
 Fatal or non-fatal hemorrhage from any tissue
or organ
 Necrosis of skin and other tissues
 Other adverse reactions reported less
frequently include:
 Systemic cholesterol microembolization
 Alopecia
 Purple toes syndrome, urticaria, dermatitis
including bullous eruptions
THROMBOSIS AND AF
 AF is the most common arrhythmia seen
in clinical practice.
 Without appropriate anticoagulant
treatment, most patients with AF are at
increased risk of cardioembolic stroke.
THROMBOSIS AND CORONARY ARTERY
DISEASE
 Cardiovascular disease is the leading cause of
death in industrialised countries. Coronary
artery disease (CAD) is the most common form
of cardiovascular disease. In CAD,
atherosclerosis damages the coronary artery
wall, predisposing to thrombus formation. The
symptoms and severity of acute coronary
syndromes (unstable angina and myocardial
infarction) vary depending on the degree to
which thrombi occlude the coronary arteries.
VASCULAR DISORDERS
 Pattern of bleeding: purpura
 Causes……
- Screening tests for hemostasis:
- BT: prolonged
- Platelet count: normal
- - PT, PTT, TT: normal
BLEEDING DISORDERS
 Abnormal bleeding may result from
- Vascular disorders
- Thrombocytopenia (  platelet count)
- Defective platelet function (qualitative
defect)
- Coagulation disorders
HEREDITARY BLEEDING DISEASES
 Von Willebrand´s disease
 Hemophilia A
 Hemophilia B
 Hemophilia C
 Factor V deficiency
 Factor VII deficiency
 Factor XIII deficiency
 Prothrombin deficiency
 Afibrinogenemia
ACQUIRED BLEEDING DISORDERS
 Consumption coagulopathies
 DIC-diseminated intravascular
coagulation
 Microangiopathic hemolytic anemia
 Vitamin K deficinecy
 Liver diseases
HEMOPHILIA A
 X-linked disorder
 Quantitative or qualitative disorder of
factor VIII
 Screening tests:
 BT: normal
 Platelet count: normal
 PT: normal
 PTT: prolonged
 Platelet count: normal
 Specific test: FVIII assay: decreased activity
(a) Factor VIII synthesis.
(b) Hemofilia A has a defect synthesis of VIIIc.
(c) von Willebrand ´s disease has a reducted synthesis
of vWF
HEMOPHILIA B
 Also called Chritmas disease
 Compared to hemophilia A:
- Less common
- same presentation
- Same screening tests results
- Specific test: FIX assay: decreased
activity
VON WILLEBRAND DISEASE
 Autosomal dominant disease
 Quantitative or qualitative disorder
of vWF
 Von Willebrand factor acts as a
carrier for FVIII
 Acts as an essential cofactor for
platelet adhesion and aggregation
VON WILLEBRAND DISEASE
 Screening tests:
 BT : prolonged.
 Platelet count: normal
 PT: normal
 PTT: prolonged
 Specific tests:
 Platelet aggregation: defective with ristocetin
 FVIII assay: decreased activity
 vWF antigen : reduced
DIC (DISSEMINATED INTRAVASCULAR
COAGULATION
 Release of tissue factor, TF.
 TF is expressed on many cell types
(endothelial, macrophages, monocytes).
 Contact with blood after damage of vessel
wall (the effects of cytokines and
endotoxins).
 TF is binding to coagulation factors which is
leading to activation of both pathways of
coagulation cascades.

DISSEMINATED INTRAVASCULAR COAGULATION
(DIC)
 Due to extensive coagulation followed
by fibrinolysis with consumption of
hemostatic factors.
 Causes:
 infection, malignancy, obstetric
complications, liver disease
DIAGNOSIS OF DIC
 BT: prolonged
 Platelet count: decreased
 PT: prolonged
 PTT: prolonged
 TT: prolonged
 Fibrinogen level: reduced
 FDPs (D dimer): increased
 Red cell fragmentation in the blood film
BT PT PTT Platelet
count
Platelet
function
Other tests
ITP P N N
Glanzman P N N N Defect
aggreg
Hemoph A N N P N FVIII assay
Hemoph B N N P N FIX assay
vWD P N P N Defect
aggreg
FVIII, vWF
DIC P P P Fibrinogen
FDPs
THANK YOU FOR YOUR ATTENTION