NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY

DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

HEMATOLOGY
Primary Hemostasis – role of blood vessels and platelets in
NORMAL HEMOSTASIS AND COAGULATION response to a vascular injury or to the commonplace desquamation
of dying or damaged endothelial cells.
Hemostasis – process that keeps circulating blood in a fluid state.
- Blood vessels contract to seal the wound or
- When an injury occurs, produces a clot to stop the reduce the blood flow (vasoconstriction).
bleeding, confines the clot to the site of injury and
dissolves the clot as wound heals. - Defects: Collagen Abnormalities,
thrombocytopenia, platelet disorders or von
- When out of balance: Hemorrhage (bleeding) or Willebrand disease.
thrombosis (clotting).
- Involves vascular intima and platelets
- Involves interaction of vasoconstriction, platelet
adhesion and coagulation enzyme activation to stop Secondary Hemostasis – activation of series of coagulation
bleeding. proteins in the plasma,
mostly serine proteases, to form a fibrin clot.

Key Cellular Elements of Hemostasis - Fibrinolysis – final event of Hemostasis,

gradual digestion and removal of the fibrin
- Cells of the vascular intima clot as healing occurs.
- Extravascular tissue factor (TF)-bearing cells
- Platelets - Involves platelets and Coagulation system.
-
Vascular Intima
Plasma Components
- Provides interface between circulating blood and the
- Coagulation body tissue.
- Fibrinolytic proteins and their inhibitors.

RODAK’S HEMOSTASIS 1
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Innermost lining of blood vessels is a monolayer of - Tissue Factor Pathway Inhibitor (TFPI) - important
metabolically active endothelial cells (EC). EC-produced anticoagulant, controls the activation of
the tissue factor pathway.
- ECs play essential roles in immune response,
vascular permeability, proliferation and hemostasis - Endothelial Protein C Receptor (EPCR) – binds
protein C and thrombomodulin catalyzes the
Anticoagulant Properties activation of the protein C pathway.

- Intact vascular endothelium prevents thrombosis by - Protein C Pathway – downregulates coagulation by

inhibiting platelet aggregation, preventing coagulation digesting activated factors V and VIII, inhibiting
activation and propagation and enhancing fibrinolysis. thrombin formation.

- Prostacyclin – platelet inhibitor and a vasodilator, - Heparan sulfate – is a glycosaminoglycan that

synthesize through the eicosanoid pathway. enhances the activity of antithrombin, a serine
protease inhibitor.
- Prevents undesirable platelet activation
in intact vessels. Procoagulant Properties

- Nitric oxide – synthesize in ECs, vascular smooth - When damaged, the vascular intima promotes
muscle cells, neutrophils and macrophages. coagulation.

- Induces smooth muscle relaxation 1. Any harmful local stimulus, whether mechanical or
and subsequent vasodilation. chemical, induces vasoconstriction in arteries and
arterioles.
- Inhibits platelet activation and
promotes angiogenesis and healthy 2. Subendothelial connective tissues of arteries and
arterioles. veins are rich in collagen, a flexible, elastic
structural protein that binds and activates
platelets.

RODAK’S HEMOSTASIS 2
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

3. ECs secrete von Willebrand factor (VWF) from - ECs support fibrinolysis, the removal of fibrin to
storage sites called Weibel-Palade Bodies when restore vessel patency, with the secretion of tissue
activated by vasoactive agents such as thrombin. plasminogen activator (TPA).

- VWF – large multimeric glycoprotein that

- During thrombus formation, both TPA and
is necessary for platelets to adhere to
plasminogen bind to polymerized fibrin.
exposed subendothelial collagen in
arterioles.
- TPA activates fibrinolysis by converting plasminogen
to plasmin, which gradually digest fibrin and restores
4. ECs secrete and coat themselves with P-selectin,
blood flow.
an adhesion molecule that promotes platelet and
leukocyte binding.
- Plasminogen activator inhibitor 1 (PAI-1) – TPA
control protein that inhibits plasmin generation and
fibrinolysis.
- ECs also secrete Ig-like adhesion
molecules called intercellular
- Thrombin-activatable fibrinolysis inhibitor (TAFI) –
adhesion molecules (ICAMs) and
Inhibitor of Plasmin generation, activated by thrombin
platelet endothelial cell adhesion
bound to EC membrane thrombomodulin.
molecules (PECAMs).
- Elevations in PAI-1 or TAFI can slow fibrinolysis and
5. Subendothelial smooth muscle cells and
increase the tendency for thrombosis.
fibroblasts support the constitutive membrane
protein tissue factor. Platelets

- Produced from the cytoplasm of bone marrow

megakaryocytes.
Fibrinolytic Properties

RODAK’S HEMOSTASIS 3
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- If injury, platelets adhere, aggregate and secrete the - During activation, ADP and Ca2+ activate
contents of their granules. phospholipase A2, converts membrane phospholipid
to arachidonic acid, Cyclooxygenase converts
- Adhesion – the property by which platelets bind arachidonic acid into prostaglandin endoperoxides.
nonplatelet surfaces such as subendothelial collagen.
- In platelet, thromboxane synthetase converts
- Importance of platelet adhesion is underscored by prostaglandin into thromboxane A2, causes Ca2+
bleeding disorders such as Bernard-Soulier released and promotes platelet aggregation and
syndrome, the platelet GP Ib/IX/V receptor is absent vasoconstriction.
and von Willebrand disease, VWF is missing or
defective. - Coagulation is initiated on tissue factor-bearing cells
(fibroblasts).
- Aggregation – the property by which platelet bind to
one another. Coagulation System

- Fibrinogen binds to GP IIb/IIIa receptors on adjacent - Plasma transports at least 16 procoagulants

platelets and joints them together in the presence of (Coagulation factors)
ionized calcium (Ca2+).

- Afibrogenemia – compromised aggregation. - All glycoproteins are synthesized in the liver. Only few
in monocytes, ECs and megakaryocytes.

- Glanzmann thrombasthenia – lack the GP IIb/IIIa - Zymogens – enzymes that circulate in an inactive
receptor. form.
- In vitro platelet aggregation, most commonly used
agonists to induce aggregation are thrombin, - Cofactors – bind, stabilize and enhance the activity of
arachidonic acid, adenosine diphosphate (ADP), respective enzymes.
collagen and epinephrine.
Classification and Function of Procoagulants

RODAK’S HEMOSTASIS 4
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- When hydrolyzed by thrombin, forms

- Plasma procoagulants may be serine proteases or fibrin clot, which is further stabilized by
cofactors except factor XIII, which is a factor XIII.
transglutaminase.
- Calcium – required for the assembly of
- Serine Proteases – proteolytic enzymes of the trypsin coagulation complexes on platelet or
family. cell membrane phospholipids.
- Synthesized as inactive
zymogen consisting of a single - Serine proteases bind to negatively
peptide chain. charged phospholipid surfaces,
predominantly phosphatidylserine,
- Activation occurs when the through + charged Ca ions.
zymogen is cleaved by the
action of another protease - Disseminated Intravascular
during the coagulation process. Coagulation (DIC) – condition when
zymogen activation is uncontrolled.
- Tissue factor – located on membranes of fibroblasts
and smooth Vitamin K-Dependent Prothrombin Group
muscles cells and soluble plasma
factors V, VIII & - Prothrombin (factor II), Factors VII, IX, and X and the
HMWK. regulatory proteins protein C, protein S and protein Z.

- Fibrinogen – ultimate substrate of the coagulation - All except proteins S and Z (cofactors) are serine
pathway. proteases when activated.

RODAK’S HEMOSTASIS 5
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Vitamin K – quinone found in green leafy vegetables

(Cauliflower, Broccoli, Asparagus, Cabbage, Avocado - Procoagulant cofactors are Tissue factor, Factor V,
and etc.). Factor VIII, & HMWK.

- Produced by intestinal organisms - Coagulation Control cofactors are Thrombomodulin,

Bacteroides fragilis and Escherichia Protein S and Z.
coli.
- Tissue factor – transmembrane receptor for factor
- In Vitamin K deficiency or in the presence of warfarin VIIa and found in extravascular cells (fibroblasts and
(Vitamin K antagonists) – Vitamin K-Dep. Smooth muscle cells). Normally, not found on blood
Procoagulants are released from the liver without the vessels ECs.
second carboxyl group added to the y carbon. - High levels in cells of the brain, lung,
placenta, heart, kidney and testes.
Coagulation Complexes
- Factors V and VIII are soluble plasma proteins. Both
Complex Components Activates
Extrinsic Tenase VIIa, Tissue IX and X are activated by thrombin and inactivated by protein
factor, C.
phospholipid and
Ca. - Factor V – glycoprotein circulating in plasma and
Intrinsic Tenase IXa, VIIIa, X
phospholipid and
present in platelet a-granules.
Ca.
Prothrombinase Xa, Va, Prothrombin - Thrombomodulin – transmembrane protein by
phospholipid and vascular ECs, is a thrombin cofactor.
Ca.

Factor VIII
Cofactors in Hemostasis

RODAK’S HEMOSTASIS 6
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Produced by ECs and megakaryocytes.

- Produced by hepatocytes.
- VWF molecules are stored in platelets a-granules and
- Free Factor VIII is unstable in plasma. Circulates in Weibel-Palade Bodies in ECs.
bound to VWF.
- VWF is decreased in Von Willebrand Disease.
- During coagulation, thrombin cleaves factor VIII from
VWF and activate. - Group O individuals have lower levels of VWF.

- Factor VIIIa binds to activated platelets and form the - VWF is an acute phase protein, as is factor VIII.
intrinsic tenase. Increase in pregnancy, trauma, infections and stress.

- Factor VIII and IX production is governed by genes

and X chromosome. Factor XI and Contact Factors
- Contact Factors (Intrinsic accessory pathway
- Hemophilia A (factor VIII) Hemophilia B (factor IX) proteins).
- Factor XII, High-molecular-weight kininogen
- Males with Hemo A have diminished factor VIII but (HMWK, Fitzgerald factor) and prekallikrein
normal VWF levels. (pre-K, Fletcher factor)

- Factor XIIa transform pre-K (glycoprotein circulates

bound to HMWK), into active form kallikrein, which
Von Willebrand Factor cleaves HMWK to form bradykinin.

- Large multimeric glycoprotein participates in platelet - HMWK is a nonenzymatic cofactor.

adhesion and transports the procoagulant factor VIII.
- Deficiencies of Factor XII, HMWK or pre-K do not
cause bleeding disorders.
RODAK’S HEMOSTASIS 7
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Fibrinogen is the primary substrate of thrombin,

- Factor XI activated by contact factor complex and by converts soluble fibrinogen to insoluble fibrin to
thrombin during coagulation from tissue factor produce a clot.
activation.
- Glycoprotein synthesized in the liver and is an acute
- Deficiencies of Factor XI (Rosenthal syndrome) phase reactant protein.

- Level increases in inflammation, infection and other

Thrombin stress conditions.

- Primary function is to cleave fibrinopeptides A & B - Platelets α-granules absorb, transport and release
from α and β chains of fibrinogen molecule, triggering abundant fibrinogen.
fibrin polymerization.
- Fibronectin – plasma protein involved in cell
- Coagulation mechanism by activating cofactors V and adhesion.
VIII and factor XI by a positive feedback mechanism.
- Plasminogen – the primary serine protease of the
- Thrombin bound to thrombomodulin activates the fibrinolytic system.
protein C pathway to suppress coagulation and it
activates TAFI to suppress fibrinolysis.
Plasma-Based (In Vitro) Coagulation: Extrinsic, Intrinsic and
- Plays a role in coagulation (fibrin), in platelet Common Pathways
activation, in coagulation control (protein C) and in
controlling fibrinolysis (TAFI). - Primary step in coagulation is the activation of factor
XII (could be found in blood, tissue factor could not)
- Reaction system begins with factor XII and culminates
Fibrinogen Structure and Fibrin Formation, Factor XIII fibrin polymerization called Intrinsic pathway.

- Coagulation factors of the intrinsic pathway. In

order of reaction

RODAK’S HEMOSTASIS 8
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

to thrombomodulin to activate the protein C

XII  pre-K  HMWK  XI  IX  VIII  X  V  control pathway and activates TAFI to inhibit
prothrombin (II)  fibrinogen fibrinolysis.

Coagulation Regulatory Mechanisms

- Tissue factor is not present in blood, tissue factor
pathway called extrinsic pathway.
Tissue Factor Pathway Inhibitor (TFPI)
- Pathway includes the factors VII, X, V, prothrombin
and fibrinogen. - Kunitz-type serine protease inhibitor and principal
regulator of the TF pathway.

Cell-Based (In Vivo, Physiologic) Coagulation - Synthesized primarily in ECs.

- Coagulation can be described as occurring in two - TFPI inhibits coagulation in a 2step process by first
phases: binding and inactivating Xa.

- Initiation – refers to extrinsic tenase complex - Protein S cofactor of activated protein C and also a
formation and generation of small amounts of factor cofactor of TFPI.
Xa, factor IXa and thrombin.

Protein C Regulatory System

- Propagation – reaction occurs on activated platelet
surface. - Revises thrombin’s function form a procoagulant
enzyme to an anticoagulant.
- COAT-platelets – partially activated by
collagen and thrombin. - Protein S – cofactor that binds and stabilizes APC.
Synthesized in the liver, 40% is free and 60% is
- Thrombin cleaves fibrinogen into a fibrin clot, bonded to complement control protein C4b-binding
activates factor XIII to stabilize the clot, binds protein (C4bBP-acute phase reactant).

RODAK’S HEMOSTASIS 9
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Systemic, accelerating hydrolysis of fibrin by bound

plasmin.
- Purpura fulminans – in neonates lack protein C.
- TPA and UPA – activators of fibrinolysis, released in
Antithrombin and Other Serine Protease Inhibitors response to inflammation and coagulation.

- Antithrombin is a serine protease inhibitor (serpin) - TPA and UPA – activate fibrin bound
binds and neutralizes serine proteases. plasminogen after thrombus formation.

- Heparin cofactor II – serpin that inactivates thrombin.

Plasminogen
- EC Heparan sulfate – natural glycosaminoglycan that
activates AT. - Plasma zymogen produced by the liver.

- ZPI – presence of cofactor (protein Z). Potent - Single-chain protein possessing five glycosylated
inhibitor of factor Xa. loops (kringles)

- Protein Z- vitamin K-dependent plasma - Kringles enable plasminogen, along with

glycoprotein in the liver. activators TPA and UPA to bind fibrin lysine
molecules during polymerization.
- Protein C Inhibitor – nonspecific, heparin-binding
serpin inhibits variety of proteases inc. APC, Plasmin
thrombin, factor Xa, factor Xia and urokinase.
- Serine protease that digest fibrin polymer by the
hydrolysis of arginine-related and lysine-related
Fibrinolysis peptide bonds.

- Final stage of coagulation, few hours after fibrin - Bound plasmin digest clots and restores
polymerization and cross linking. blood vessel patency.

RODAK’S HEMOSTASIS 10
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Free plasmin is capable of digesting plasma

fibrinogen, factor V, factor VIII and Plasminogen Activator Inhibitor 1 (PAI-1)
fibronectin.
- Inhibitor of plasminogen activation, inactivating both
TPA and UPA, preventing them from converting
PLASMINOGEN ACTIVATION plasminogen to plasmin.

Tissue Plasminogen Activator (TPA) - Single-chain glycoprotein serine protease inhibitor

produced by ECs, megakaryocytes, Smooth muscle
- ECs secrete TP, which hydrolyzes fibrin-bound cells, fibroblasts, monocytes, adipocytes,
plasminogen and initiates fibrinolysis. hepatocytes.

- With two glycosylated kringle regions, forms covalent - An acute phase reactant increased in metabolic
lysine bonds with fibrin and localizes at the surface of syndrome, obesity, atherosclerosis, sepsis and
the thrombus with plasminogen and begins digestion stroke.
process by converting plasminogen to plasmin.

Urokinase Plasminogen Activator (UPA)

α2 – Antiplasmin
- Secreted by urinary tract epithelial cells, monocytes
and macrophages. - Synthesize in the liver and inhibitor of free plasmin.

- UPA circulates in plasma at 2 to 4 ng/mL - Serine protease that have both N and C-terminal
concentration. Mix of fibrin-bound plasminogen and extension.
TPA at the time of thrombus formation.
- During thrombus formation, N-terminus covalently
linked to fibrin by factor XIIIa and C-terminal contains
CONTROL OF FIBRINOLYSIS lysine, capable of reacting with lysine-binding kringles
of plasmin.

RODAK’S HEMOSTASIS 11
NOTRE DAME OF MARBEL UNIVERSITY MEDICAL TECHNOLOGY
DEPARTMENT
REVIEW IN CLINICAL HEMATOLOGY RYAN M. PEDREGOSA, RMT

- Fragments X, Y, D and E produced by digestion of

- AP with C-terminal lysine slows fibrinolysis by either fibrin and fibrinogen by plasmin.
competing with lysine residues for plasminogen
binding and by binding directly to plasmin and - D-dimer specific product of digestion of cross-linked
inactivating it. fibrin.

- D-dimer is detectable by monoclonal antibody for D-

Thombin-Activatable Fibrinolysis Inhibitor dimer antigen.

- Plasma procarboxypeptidase synthesized in the liver - D-dimer immunoassay used to identify chronic and
activated by thrombin-thrombomodulin complex. acute DIC and to rule out venous thromboembolism in
suspected cases of deep venous thrombosis or
- Activated TAFI functions as an antifibrinolytic enzyme. pulmonary embolism.

- Inhibits fibrinolysis by cleaving exposed carboxy-

terminal lysine residues from degraded fibrin, SOURCE:
preventing binding of TPA and plasminogen to fibrin
and blocking the formation of plasmin. Rodak’s Clinical Hematology Principles and Applications. 5 th edition

- TAFI play a role in regulating inflammation and wound

healing.

Fibrin Degradation Products and D-Dimer

- Plasmin cleaves fibrin and produces a series of fibrin

fragments: X, Y, D, E and D-D.

RODAK’S HEMOSTASIS 12