FIBRINOLYSIS

Once fibrin is no longer needed, the fibrinolytic

system is activated, converting fibrin to its soluble
degradation products through the action of plasmin.

In physiologic conditions, fibrinolysis is precisely

regulated by the measured participation of
activators, inhibitors and cofactors.
FIBRINOLYSIS
COMPONENTS OF THE
FIBRINOLYTIC SYSTEM

Plasminogen
Plasminogen Activators
Plasmin
Inhibitors of Fibrinolysis
PLASMINOGEN

 Synthesized primarily in the liver, plasminogen is

a single-chain proenzyme.
 The plasminogen gene is located on chromosome 6.

 Synthesis of plasminogen is amplified by IL 6.

 The activation of plasminogen to plasmin results

from the cleavage of a single Arg-Val peptide bond
at position 561-562.
 PLASMINOGEN ACTIVATORS
a) t-PA (tissue plasminogen activator)
 t-PA is synthesized and secreted primarily by endothelial cells as
single chain t-PA and then activated in the presence of plasmin
and kalicrein into two chains t-PA.
 in the presence of fibrin a dramatic increase in affinity (100 x)
between t-PA and its substrate (plasminogen) takes place;
therefore, fibrin is in fact the most important trigger of fibrinolysis.
 t-PA appears to represent the major circulating activator of
plasminogen.
b) u-PA (urokinase plasminogen activator)
 This activator was first identified in the urine. It was subsequently
detected in the media of cultured human kidney cells,
endothelial cells, malignant cells.
 u-PA is synthesized as single chain u-PA (prourokinase) and
activated by plasmin and kalicrein in two chain u-PA (active form).
 u-PA is an effective plasminogen activator both in the presence
and in the absence of fibrin.
PLASMIN

 Plasmin is a serine protease resulted from the

activation of plasminogen with proteolytic effect
on fibrinogen and fibrin as well as on FV,
FVIII, FXIII and fvW.
 Plasmin plays also a role in tissue remodeling
and healing (activates TGFβ).
FIBRINOGEN STRUCTURE

 Fibrinogen consists of three pairs of polypeptide

chains: Aα, Bβ and γ.
 These are linked together by disulphide bonds in such
a way that N-terminal regions of the 6-polypeptide
chains meet to form a central E-domain.
 The C-terminal regions form the D-domain and
these are joined by α-helical ropes to the central E-
domain to give the characteristic fibrinogen structure.
 Thrombin (IIa) cleaves the two short
peptides from the N-terminal regions
of the Aα and Bβ chains - these
peptides are known as
Fibrinopeptide A (FpA - 16 amino
acids) and Fibrinopeptide B (FpB -
14 amino acids) respectively.
 The removal of the N-terminal
sequences from the Aα and Bβ chains
chains reveals new N-terminal
sequences in the Aα and Bβ chains
located within the E domain, known as
'knobs'.
 These knobs can interact
spontaneously with the D-domains
of other fibrin fibers to form fibrin
polymers.
 Under the influence of factor XIIIa,
cross-linking of these fibrin polymers
occurs to form cross-linked fibrin
 DEGRADATION OF FIBRINOGEN: FDPS PRODUCTION
 Plasmin cleavage of fibrinogen produces
carboxyl-terminal fragments from the D
domains of fibrinogen (Aα fragment and
Bβ fragment).
 The resulting molecule is termed
fragment X and it represents a clotable
form of fibrinogen.
 In a series of subsequent reactions,
plasmin cleaves the three polypeptide
chains that connect the D and E domains,
giving rise to free D domain plus the
binodular D-E fragment known as
fragment Y.
 Finally, domains D and E are separated
from each other.
 Although fragment X can be converted to
fibrin by thrombin, the fragments Y, D,
and E are all nonclotable and in fact may
inhibit the spontaneous polymerization of
fibrin.
DEGRADATION OF FIBRIN

 Plasmin degradation of fibrin

leads to a distinct set of
molecular products.
 Species similar to fragments Y,
D, and E but lacking
fibrinopeptides (A and B) sites
are released from non–cross-
linked fibrin.
 If fibrin has been extensively
cross-linked by factor XIII,
however, the resulting D
fragments are cross-linked
to an E domain fragment
and they are called D
dimers.
INHIBITORS OF FIBRINOLYSIS

a) Plasminogen Activator Inhibitors (PAI)

b) Plasmin inhibitors
c) Thrombin-Activable Fibrinolysis Inhibitor - TAFI
a) PLASMINOGEN ACTIVATOR
INHIBITORS

 PAI-1 (plasminogen activator inhibitor 1)

 PAI-1 is the most important and rapidly acting
physiologic inhibitor of both t-PA and u-PA.
 PAI-1 is released by endothelial cells, platelets,
monocytes, macrophages, hepatocytes, adipocytes and
others.
 PAI-2 (plasminogen activator inhibitor 2)
 PAI-2 inhibits both two-chain t-PA and two-chain u-PA;
 Originally purified from human placenta.
b) PLASMIN INHIBITORS

 The action of plasmin is negatively modulated by a

family of serine protease inhibitors, called serpins
which have a common mechanism of action by forming
an irreversible complex with the target protease
following proteolytic cleavage of the inhibitor by the
target protease.
 Within this complex, both the protease and the
inhibitor lose their activity.
 α2 antiplasmin (α2-AP) = is produced by the liver but α2-
AP is also a constituent of platelet α granules; Plasmin
released into blood flow or in the vicinity of a platelet-rich
thrombus is immediately neutralized by α2-AP; α2-AP is
the most important plasmin inhibitor.
 α2 macroglobulin – inhibits plasmin with approximately 10
percent of the efficiency exhibited by α2-AP.
 α1 antitrypsin.
 c) TAFI = THROMBIN-ACTIVABLE
FIBRINOLYSIS INHIBITOR

 TAFI is a plasma carboxypeptidase with specificity for

carboxyl-terminal arginine and lysine residues of
fibrin preventing plasminogen binding to fibrin and
therefore plasminogen activation to plasmin, t-PA
mediated.
 TAFI acts as a potent inhibitor of fibrinolysis.

 TAFI undergoes limited proteolysis in the presence of

thrombin, which leads to its activation.
FIBRINOLYSIS
THROMBOLYTIC AGENTS
 Thrombolytic agents are serine proteases that work by
converting plasminogen to the natural fibrinolytic
agent plasmin.
 The goal of thrombolytic therapy is to reinstate the
blood flow throw the occluded blood vessels.
 t-PA: alteplase, reteplase, and tenecteplase.
 urokinase
 streptokinase - antigenic problems
 APSAC (anisoylated plasminogen streptokinase activator
complex) - antigenic problems
 Thrombolytic agents are indicated for the treatment of
acute myocardial infarction, acute ischemic stroke,
acute massive pulmonary embolism, deep vein
thrombosis and arterial thrombosis.
FIBRINOLYTIC ASSAYS

1. Euglobulin Clot Lysis Time (ECLT)

2. Fibrin(ogen) Degradation Products assay
3. D Dimers assay
 1. EUGLOBULIN CLOT LYSIS TIME (ECLT)
 Reflects the overall fibrinolytic activity of plasma.
 The patients poor platelets plasma (PPP) sample is diluted with acetic
acid.
 A precipitate forms (the euglobulin fraction of plasma) which contains
plasminogen, plasminogen activators (primarily t-PA) and fibrinogen.
 The precipitate is dissolved in buffer and then is clotted with thrombin
and the time to clot lysis is determined by inspection every 15 minutes.
 Reference Range: 90-240 minutes
 Hyperfibrinolysis: <60 minutes
 Primary fibrinolysis (liver disease)
 Disseminated intravascular coagulation
 Deep Venous thrombosis
 Acute myocardial infarction
 Pulmonary embolism
 Thrombolytic therapy
 Post-surgery
2. FIBRIN(OGEN) DEGRADATION PRODUCTS
ASSAY

 latex-agglutination or ELISA
 normal values < 10 microg/ml
 FDPs are elevated in:
 Primary fibrinolysis (liver disease)
 Disseminated intravascular coagulation (DIC)
 Deep Venous thrombosis
 Acute myocardial infarction
 Pulmonary embolism
 Thrombolytic therapy
 Post-surgery
3. D DIMERS ASSAY

 D dimers are generated when cross-linked fibrin is

degraded and so they are not generated if non-cross
linked fibrin or fibrinogen is broken down - they are,
therefore, fundamentally different from FDPs
 ELISA: normal values = 0.25–0.75 microg/ml
 D-dimers are raised in:
 Disseminated Intravascular Coagulation
 Deep Venous Thrombosis
 Acute myocardial infarction
 Pulmonary embolism
 Thrombolytic therapy
 Post-surgery
PRIMARY FIBRINOLYSIS

 Primary fibrinolysis or fibrinogenolysis, to be more

exact, occurs when plasmin is generated in the
absence of coagulation (fibrinolysis is not activated
by the fibrin clot).
 This has been described in liver failure (acquired
deficiency of α2-antiplasmin), prostatic carcinoma,
and rare cases of hereditary deficiency of α2-
antiplasmin or hereditary deficiency of PAI-1.

 Primary fibrinolysis can be distinguished from DIC (a

form of secondary fibrinolysis) by finding greatly
elevated fibrinogen degradation products while
D-dimer levels are normal.
DISSEMINATED INTRAVASCULAR COAGULATION (DIC)
 Disseminated intravascular coagulation (DIC) is a clinico-
pathologic syndrome in which widespread intravascular
coagulation is induced by procoagulants that are introduced or
produced in the blood circulation and overcome the natural
anticoagulant mechanisms.
 Tissue factor exposure to blood is the most common
trigger of DIC.
 endothelial cells generate and express tissue factor during the
systemic inflammatory response syndrome (e.g., gram-
negative and gram-positive infections, fungemia, burns, severe
trauma).
 contact between blood and tissue factor constitutively
present on membranes of cells foreign to blood (e.g.,
malignant cells, placenta, brain cells, adventitia, traumatized
tissues).

 DIC determines multiorgan dysfunction caused by

microthrombi and bleedings caused by consumption of platelets
and coagulation factors, as well as secondary fibrinolysis .
 Both DIC and the underlying disorders causing DIC contribute
PATHOPHYSIOLOGY
DIC - LABORATORY FEATURES

 Thrombocytopenia,
 microangiopathic hemolytic anemia;
 leukocytosis
 APTT, PT, TT, TR are prolonged (due to consumption
of the coagulation factors)
 Fibrinogen <150 mg/dl
 FDPs and D-dimers values are elevated

 The underlined tests constitute the DIC panel.

 TREATMENT:

 Early detection
 Aggressive treatment of the underlying disorders:
 intensive antibiotic treatment in patients with gram-negative bacteremia
 hysterectomy in patients with abruptio placentae
 debridement of crushed tissues
 Intensive support of vital functions is required:
 Volume replacement and correction of hypotension and oxygenation.
 Careful monitoring of pulmonary, cardiac and renal function enables
prompt institution of supportive measures, such as:
 use of a respirator for better oxygenation
 inotropic drugs for improvement of cardiac output
 maintenance of the electrolyte balance.
 Platelet concentrates, cryoprecipitate and fresh-frozen plasma replace
the hemostatic factors and inhibitors of blood coagulation commonly
depleted
 Heparin in selected patients.
 AT-III concentrate infusion.
 Test Patient values Normal values Units
Hb 7.6 11 - 16 g/dl
Ht 28.5% 36-46 %
Red Blood Cells 2.4∙10⁶ 4∙10⁶ - 5.5∙10⁶/mmc
Reticulocytes 1.7% 0.5 – 1.5%
MCV 88 80 – 100 µmc(fl)
MCHC 33.2 32 – 36 g/dl E
White Blood Cells 15.554 4.000 – 10.000/mmc
Platelets 69.000 150.000 - 400.000/mmc
Bleeding Time 16 6-10 minutes
APTT 87 25 - 35 seconds
INR 3.5 0.9 – 1.2
FDPs 60 <10 µg/ml
D dimers 3,5 0.22 – 0.74 µg/ml

Fibrinogen 97 200 - 400 mg/dl