7 Coagulation and Fibrinolysis

Key Points

Physiologic hemostasis consists of the plasma

coagulation, fibrinolysis, and anticoagulation protein
systems.

Physiologic hemostasis is initiated by factor VIIa and

tissue factor.

Physiologic hemostasis is not fully represented by

current assays to detect coagulation abnormalities
such as aPTT and PT.

Current assays to assess coagulation protein

abnormalities have good diagnostic power to
recognize specific defects in coagulation proteins.

Acquired coagulation protein defects more commonly

reflect general medical disorders than specific protein
defects.

As soon as vessel is injured, the following events are

observed:
1. tissue factor is upregulated
2. TF forms complex with factor 7a which
activates respectively:
a. 9 (which in turn activates)
b. 10
3. Factor 10a in the presence of factor 5a,
activates factor 2(prothrombin) to factor 2a
(thrombin), the major clotting enzyme

History of the Coagulation Cascade

1964 = Ratnoff and MacFarland; Davies proposed the

coagulation cascade starting with factor 12 to fibrin
formation; Ratnoff (1955) earlier published that
factor 12 deficiency was not associated with bleeding

Mid 1970s = cofactors for factor 12 activation

(prekallikrein; high molecular weight kininogen) were
not associated with bleeding state

1977 = Osterud and Rappaport: factor 7a is able to

activate factor 10

2003 = Boze: factor 7a and tissue factor complex can

not directly activate factor 10 but has to go thru
factor 9 activation

At present, it is believed that there is no intrinsic

pathway(this occurs only in the test tube) and the
initiator for physiologic hemostasis is factor 7a and
tissue factor complex
Physiologic hemostasis has two parts:

Cellular component = platelets, endothelial cells,

neutrophils and monocytes

Plasma proteins producing clot, dissolution of clot

(fibrinolytic system) and the naturally occurring serine
protease inhibitors that terminate activities of
coagulation and fibrinolytic systems
Endothelium and Platelets

Constitutive anticoagulation of the endothelium:

1. Glycosaminoglycans that bind antithrombin
2. Thrombomodulin = locus where protein C is
activated by low levels of thrombin
3. Ectonucleotidase (CD 39) = degrades ADP
4. Prostacyclin and nitric oxide= prevents
platelet activation
5. Bind plasminogen, tissue plasminogen
activator, urokinase= contribute to
fibrinolysis
Events after vessel injury

Collagen exposed platelets adhere to site of injury

vWF binds platelets to subendothelium
activation of platelets degranulation of platelets
platelets aggregate temporary plug

Coagulation Protein System

Two types: zymogens and cofactors

Zymogens: phospholipid bound(Vit K dependent) and

surface dependent pro enzymes

Vit K dependent factors undergo carboxylation of

their amino terminals before they bound to
phosphoplipid and only then, become functional

Cofactors: receptors for coagulation proteins

Proteins of the Coagulation System
Surface bound
Vitamin K-dependent
Factor 12
Factor 7
Pre-kallikrein
Factor 9
Factor 10
Factor 11
Factor 2
Protein C

Co-factors
HMWKininogen
Factor 8
Factor 5
Fibrinogen
Protein S

Physiologic Protein Assemblies

1. Tenase: Factor 9a, thrombin activated Factor 8 on
phospholipid surfaces or cell membranes in an
ordered structure with Factor 10 to activate Factor
10a.
2. Prothrombinase: assembly of Factor 10a and thrombin
-activated Factor 5a on phospholipid membranes or
cell membranes in an ordered structure with Factor 2
(prothrombin) to accelerate its activation to
Factor 2a (thrombin).
rainwater@mymelody.com || 1st semester, AY 2011-2012

Formation of fibrin and the fibrinolytic system

Fibrin:
Fibrinogen has central E domain and D terminals
thrombin cleaves fibrinopeptides A and B in the E
domain soluble fibrin monomer these assemble
with end to end and side to side association to form a
non-covalent fibrin polymer activated Factor 13
crosslinks them into an insoluble fibrin clot

Plasmin cleaved soluble fibrinogen or fibrin

Plasmin cleaves alpha chains from the D terminals,

making fragment XFragment X is asymmetrically
cleaved into Fragment D and Fragment Y Fragment
Y is further cleaved by plasmin into Fragment D and E
Plasmin cleaved insoluble, cross-linked fibrin

When insoluble, cross-linked fibrin is proteolyzed by

plasmin, the neo-epitope between the D domains is
preserved and the liberated fragment consists of the
D dimer together with an E domain
Anticoagulant System

Current hypothesis for the initiation of the Hemostatic System

TF+F7a activates F9; F9a activates F10 (maybe

inhibited by TFPI); F10a converts prothrombin to
thrombin which converts fibrinogen to fibrin; fibrin
stimulates plasmin which is inhibited by thrombinactivatable fibrinolysis inhibitor to decrease lysis
activity

Thrombin accelerates convertion of F11 to F11a

which accelerates F10 to F10a which acts on
prothrombin
Approach to patients with hemostatic problem
1. Rule out initially acquired coagulation protein
deficiency
2. Coagulation factor prolongation may be due to:
a. True protein deficiency
b. Inhibitor(Ig or abnormal production of
heparin, fibrinectin, cryoglobulin)
c. Abnormal coagulation protein
d. Enhanced clearance (Ag-Ab complex)
Review History
Primary
Onset after trauma: Spontaneous
Site:
Skin, mucous
Clinical Examples:
Thrombocytopenia
Platelet defect
vWD; scurvy

Secondary
Delayed
Deep tissues
Factor deficiency
Liver disease
Acquired inhibitors

Anticoagulation Protein Systems

Protein C and S

Activated Protein C:
1. Decreases thrombin formation =
inactivates factors 5a and 8a
2. Stimulates fibrinolysis = liberates tP
activator upon binding with ECPR which
activates PAR (protease activated receptor)

Protein S (co-factor)
o Free and bound (free form is the receptor)
Plasma serine protease inhibitor system
rainwater@mymelody.com || 1st semester, AY 2011-2012

Physiologic hemostasis vs. Clinical Assays

Dictum: physiologic hemostasis is initiated by

upregulation of TF + Factor 7a; NO clinical assay can

assess this event
PT and aPTT monitor late event of actual clot

formation; neither tests represent physiologic

hemostasis

PT = addition of excess TF creates a very

unphysiologic change in the normal stoichiometric
relationship of factors, while aPTT measures more
proteins than necessary for physiologic hemostasis
Activated PTT
Negatively charged surface + phospholipid + plasma

with 3.2 g% sodium citrate incubate

Add calcium chloride

Measure clotting time

A study of the (supposedly) intrinsic factors and the

common pathway factors

PT

Tissue thromboplastin(animal derived or

recombinant; tissue factor plus exogenous
phospholipid) + plasma incubate
Add 30 mM calcium chloride
Measure clotting time
Evaluates extrinsic and common pathway

Thrombin time/Reptilase time

Purified exogenous thrombin + plasma

Measure clotting time

Measures fibrinogen

Prolonged in: hypofibrinogenemia; dysfibrogenemia

Interpretation of abnormal coagulation tests

Any defect below a specific point will lead to an

abnormal result above the defect

E.g.:
1. Inhibitor of factor 8: abn factor 11 assay
2. Deficient/abnormal fibrinogen: affect
results of all clotting tests
Abnormal aPTT alone

Associated with bleeding: factors 8, 9, 11 defects

Not associated with bleeding: factor 12, prekallikrein,

high molecular weight kininogen, lupus anticoagulant

Abnormal PT alone and combined abnormal aPTT and PT
Abnormal PT alone: factor 7 defects

Combined:
1. Medical conditions: anticoagulation, DIC,
liver disease, vitamin K deficiency, massive
transfusion
2. Rare: dysfibrinogenemias, factors 10, 5, 2
defects
Rare bleeding disorders (not picked by tests of hemostasis)

In order of frequency: Factor 13 defects, alpha-2

antiplasmin defects, plasminogen activator inhibitor/
defects, alpha-1 antitrypsin (Pittsburg)
If your patient has an inhibitor:

Do mixing studies using the specific factor inhibitor

assay or the effect on PT or PTT

For e.g., inhibitor for factor 8

Mixing normal and patients plasma should have
lower value in the factor assay of factor 8 than the
calculated expected value for the dilution.

Using PT, mix normal plasma and patients plasma

Normalization of value excludes inhibitor but

prolongation is suggestive of an inhibitor
rainwater@mymelody.com || 1st semester, AY 2011-2012

The acquired coagulation protein deficiencies are common

than hereditary deficiencies
Hereditary deficiencies: Henrys, pp 737-742

In practice, consider in your patients the following

common acquired coagulation protein deficiencies:

anticoagulation, DIC, liver disease, Vitamin K
deficiency, massive transfusion effect and inhibitors
Disseminated intravascular coagulation

Pathophysiology: activation of the coagulation and

fibrinolytic system simultaneous formation of
thrombin and plasmin consumption of coagulation
factors and inhibitors

Causes: sepsis, malignancy, OB complications,

massive tissue necrosis

Two phenotypes:
Hyperfibrinolytic state: prolonged PT and
1.
PTT; decreased fibrinogen and platelet
counts.
2.
Prothrombotic state: normal PT and PTT;
mildly decreased platelets, normal or
increased fibrinogen
Confirmatory: D-dimer

D-dimer can be positive also in resolving large vessel

thrombosis and soft tissue hematomas
Liver Disease

Serious liver diseases prolong PT and PTT

Reasons: decreased synthesis(all Vitamin K-dependent

factors 2, 7, 9, 10, protein C and S, protein Z)
Usually with dysfibrinogenemia

st

Prekallikrein 1 protein to decrease in liver disease

Fibrinogen last protein to decrease in liver disease

Vitamin K deficiency

After 4-6 weeks of antibiotics and parenteral nutrition

in the very ill patient

Also seen in anatomic bypass of the small intestines,

malabsorption, biliary tract obstruction, warfarin,
alcoholics
Massive transfusion
Replacement of >1.5 blood volume in 24 hours

Bleeding due to dilution of clotting factors, DIC or

acquired platelet dysfunction

Lab DX: prolonged PT and PTT, decreased fibrinogen,

decreased platelets

In a 70kgs patient given 1.5x his blood volume, the

circulating anticoagulant can be 735 ml
Acquired Coagulation Protein Inhibitors
1. Factor 8 inhibitor = most common; prolonged aPTT;
usually seen in the elderly with B cell malignancy,
connective tissue disease, post=partum
2. Amyloidosis = with decreased factor 10 and 9 as a
result of absorption of coagulation proteins onto
amyloid protein
3. Hypergammaglobulinemia = with pan-inhibitors(
multiple myeloma and Waldenstroms
macroglobulinemia) and usually with
dysfibrinogenemia
4. Lupus anticoagulant or Phospholipid antibody
(thrombotic) = antibodies directed to epitopes of
proteins bound to phospholipids

rainwater@mymelody.com || 1st semester, AY 2011-2012