HEMOSTASIS

●​ To limit blood loss from an injured vessel

●​ Functions:
○​ Prevents blood loss
○​ Tissue repair
○​ Removes fibrin clot
●​ Endothelial cells - found in lining of interior of blood and lymphatic vessels
○​ Regulate perfusion (blood flowing through body tissues and organs),
permeability, local coagulation, and immune responses
■​ Modulate contraction and relaxation of underlying smooth muscle
maintaining proper circulation
■​ Controls exchange of nutrients, gases, and waste by regulating
permeability
■​ Release promoters or inhibitors of clotting, balancing regulation of
coagulation
○​ Prevent clotting by providing non-adhesive surfaces
○​ Inhibit coagulation process - produces anticoagulants
○​ Hemostasis - promote formation of temporary clot
○​ Inflammation - regulation of adhesion and migration to sites of injury or infection
by expression of adhesion molecules such as selectins and integrins
○​ Repair - proliferate and migrate to undergo angiogenesis
○​ Injury - due to endothelial dysfuncion
●​ Products which inhibit coagulation process
○​ Heparan sulfate - cofactor for antithrombin III, reducing thrombin activity and
inhibiting clot formation
○​ Thrombomodulin - found on the surface of endothelial cells. If thrombin +
thrombomodulin, it undergoes conformational change to activate protein C >
inactivate FVa and VIIa with cofactor protein S
○​ Prostacyclin (PGI2) - inhibits platelet aggregation and vasodilation; increases
cAMP in platelets > protein kinase A inhibits release of calcium ions
○​ Nitric oxide - produced by endothelial cells by nitric oxide synthase; potent
vasodilator, inhibits platelet aggregation > increased cGMP > regulate BP and
increase blood flow
●​ 4 major physiologic events:
1.​ Vascular phase: Vascular constriction
2.​ Platelet phase: Platelet plug formation
3.​ Coagulation phase: Fibrin formation
4.​ Fibrinolysis

Constriction > Platelet plug > Fibrin > Fibrinolysis

Vascular Constriction
●​ Initial response to injury
○​ More pronounced in vessels with medial smooth muscle
○​ Dependent on local contraction of smooth muscle
●​ Begins prior to platelet adherence as reflex response to various stimuli
●​ Mediators:
1.​ Thromboxane A2 - produced locally at site of injury via release of arachidonic
acid from platelet membranes
➔​ Potent constrictor of smooth muscle
2.​ Serotonin (5-hydroxy-tryptamine / 5-HT) - released during platelet aggregation
➔​ Potent vasoconstrictors
3.​ Endothelin - synthesized by injured endothelium
4.​ Bradykinin
5.​ Fibrinopeptides
➔​ Involved in coagulation schema
➔​ Capable of contracting vascular smooth muscle

Arachidonic acid Arachidonic acid

↓ released from platelet membranes ↓ shuttled to adjacent endothelial cells
Cyclooxygenase Prostacyclin (PGI2)
↓ ●​ Vasodilator
Prostaglandin G2 (PGG2) ●​ Acts to inhibit platelet aggregation
↓
Prostaglandin H2 (PGH2)
↓
Thromboxane A2 (TXA2)
●​ Potent vasoconstrictor
●​ Has platelet aggregation effects

●​ Extent of vasoconstriction varies with degree of vessel injury

○​ E.g. small artery with lateral incision may remain open due to physical forces
Similar-sized vessel completely transected may contract to extent that
bleeding ceases spontaneously

Platelet Function
Platelets (150 000 - 400 000/uL)
●​ Anucleate fragments of megakaryocytes
●​ ~30% circulating platelets may be sequestered in the spleen
○​ If not consumed in clotting reaction, platelets are normally removed by spleen
●​ Average life span of 7-10 days
●​ Role in Hemostasis:
○​ Formation of hemostatic plug
○​ Thrombin formation
●​ Do not normally adhere to each other or to vessel wall
 ●​ Can form a plug = aids in cessation of bleeding when vascular disruption occurs
●​ Thrombopoietin: predominant mediator of platelet function

Primary Hemostasis
1.​ Injury to intimal layer in vascular wall
2.​ Exposes subendothelial collagen
3.​ Adhesion
○​ Platelets adhere to exposed subendothelial collagen
■​ Requires vWF: binds to GP I/IX/V on platelet membrane
4.​ Activation
○​ Platelets initiate release reaction for recruitment of other platelets from circulating
blood to seal disrupted vessel (primary hemostasis)
5.​ Aggregation
○​ Reversible
○​ Principal mediators: adenosine diphosphate (ADP), serotonin (5-HT)
○​ Cyclooxygenase
■​ Irreversibly inhibited by aspirin
■​ Reversibly blocked by NSAIDs
■​ Not affected by COX-2 inhibitors
○​ Second wave:
■​ Release reaction occurs in which substances are discharged: ADP, Ca2+.
Serotonin, TXA2, a-granule proteins
■​ Fibrinogen is required cofactor: bridge for GP IIb/IIIa on activated
platelets
■​ Results in compaction of platelets into a plug (irreversible)
■​ Thrombospondin - secreted by a-granule: stabilizes fibrinogen binding to
activated platelet surface, strengthens platelet-platelet interactions
■​ Platelet Factor 4 (PF4) and a-thromboglobulin are secreted
■​ Potent heparin antagonist
■​ Inhibited by aspirin, NSAIDS, cAMP, and nitric oxide
■​ Alterations occur in phospholipids of platelet membrane that allow calcium
and clotting factors to bind on platelet surface
■​ Altered lipoprotein surface (PF3) catalyze reactions involved in:
●​ Conversion of prothrombin (II) to thrombin (IIa) by
activated Xa in presence of V and calcium
●​ Activated factor IX (IXa), factor VIII and calcium activated
factor X
■​ Plays a role in initial activation of factors XI and XII
 Primary Hemostasis Thrombin Formation

●​ Hemostatic plug ●​ Amorphous plug

●​ Reversible ●​ Irreversible
●​ Not affected by heparin administration ●​ Involves fibrinogen-dependent
●​ Triggered by injury to vessel wall degranulation
●​ Requires vWF ●​ Seals the injury
●​ Platelets adhere to subendothelial ●​ Requires fibrinogen
collagen of disrupted vascular tissue ●​ Release reaction discharges
●​ Platelets initiate a release reaction substances
that recruits other platelets to seal
disrupted vessel
●​ Platelets aggregate

Coagulation
1.​ Intrinsic Pathway - intrinsic to circulating plasma; no surface required to initiate process
12 > 11 > 9 > 7
2.​ Extrinsic Pathway
TF released or exposed on surface of endothelium + circulating factor 7 (3 + 7) > 7a
3.​ Common Pathway
8a > 10 → 10a → 5a > 2 (prothrombin) > thrombin; 1 (fibrinogen) > fibrin

●​ Clot formation occurs after fibrin monomers are cross-linked to polymers with
assistance of FXIII (FSF)
●​ Phases of Coagulation
1.​ Initiation
a.​ Primary pathway is initiated by TF exposure following
subendothelial injury
b.​ TF + 7a
c.​ Activation of FX → Xa and FIX → IXa
d.​ Activation of FV → Va
e.​ Prothrombinase complex - generates small amounts of thrombin
from prothrombin in calcium-dependent process
2.​ Amplification
a.​ Platelets adhere to ECM components at site of injury
b.​ Becomes activated upon exposure to thrombin and other stimuli
3.​ Propagation
a.​ Tenase (IF) complex (F8a/9a) and prothrombinase (EF) complex
(F5a/10a) are assembled on surfaces of activated platelets
b.​ Results in large-scale generation of thrombin (thrombin burst) and
fibrin
●​ Intrinsic factor complex: F8a/9a
○​ Factor 9a - responsible for bulk conversion of Factor 10 to 10a
○​ 50x more effective at catalyzing factor 10 activation than extrinsic
complex
 ○​ 5-6 orders of magnitude more effective han factor 9a alone
●​ Once formed, thrombin leaves membrane surface and converts fibrinnogen by 2
cleavage steps into:
○​ Fibrin
○​ Fibrinopeptide A
■​ Removal permits end-to-end polymerization of fibrin molecules
○​ Fibrinopeptide B
■​ Cleavage allows side-to-side polymerization of fibrin clot
■​ Facilitated by TAFI (Thrombin-activatable fibrinolysis inhibitor) -
acts to stabilize resultant clot
●​ Ways to prevent propagation of clot beyond the site of injury
1.​ Feedback inhibition on coagulation cascade deactivates enzyme
complexes → thrombin formation
a.​ Thrombomodulin presented by endothelium serves as thrombin
sink by forming complex with thrombin to render it no longer
available to cleave fibrinogen
b.​ Activation of protein C reduces further thrombin generation by
inhibiting factors 5, 8
2.​ Tissue plasminogen activator (tPA) is released from endothelium following
injury → cleavage of plasminogen to initiate fibrinolysis
a.​ APC consumes PAI-1 ( plasminogen activator inhibitor-1) →
INCREASED tPA activity and fibrinolysis
b.​ TFPI (tissue factor pathway inhibitor) is released to build on
anticoagulant response to inhibit thrombin formation → blocks
TF-VIIa complex → reduce production of F10a, F9a
c.​ AT-III (Antithrombin-III) neutralizes all procoagulant serine
proteases and inhibits TF-VIIa complex
●​ Most potent mechanism of thrombin inhibition: APC system
○​ APC + protein S on phospholipid surface
■​ Cleaves F5a and F8a → cannot participate in formation of TF-7a
or prothrombinase complexes
●​ FACTOR V LEIDEN: F5 is resistant to cleavage by APC,
remaining active as procoagulant
○​ Predisposed to venous thromboembolic events
●​ Plasmin: serine protease from proenzyme plasminogen that degrades fibrin clot
○​ tPA: Main circulating form
■​ Made by endothelium and other cells of the vascular wall
■​ Selective for fibrin-bound plasminogen
■​ Endogenous fibrinolytic activity occurs predominately at site of clot
formation
○​ uPA (urokinase plasminogen activator)
■​ Produced by endothelial cells and urothelium
■​ Not selective for fibrin-bound plasminogen
●​ Thrombin-TM complex: activates TAFI
 ○​ Leads to mixed effect on clot stability
○​ Inhibits fibrinolysis directly
○​ Removal of terminal lysine on fibrin molecule renders clot more
susceptible to lysis by plasmin

aPTT PT Vitamin K deficiency

Warfarin use
Intrinsic Extrinsic

2, 9, 10, 11, 12 2, 7, 10 2, 7, 9, 10
Fibrinolysis
●​ Allows restoration of blood flow during the healing process following injury
●​ Begins at the same time clot formation is initiated
1.​ Fibrin polymers degraded by plasmin
2.​ Plasminogen is converted to plasmin by several plasminogen activators: tPA
3.​ Plasmin degrades fibrin mesh
4.​ Production of circulating fragments or fibrin degradation products
5.​ Clearance by other proteases or by kidney and liver: directed by circulating
kinases, tissue activators, kallikrein
6.​ tPA synthesized by endothelial cells, released by cells on thrombin stimulation
7.​ Bradykinin cleaved from HMWK by kallikrein: enhance release of tPA
8.​ Both tPA and plasminogen bind to fibrin
9.​ Trimolecular complex cleaves fibrin very efficiently
●​ Kept in check through mechanisms such as:
○​ tPA activates plasminogen more efficiently when bound to fibrin
■​ Plasmin is formed selectively on the clot
○​ Plasmin is inhibited by a2-antiplasmin
■​ A2-antiplasmin is cross-linked to fibrin by FSF
■​ Ensures clot lysis does not occur too quickly
○​ Any circulating plasmin is inhibited by a2-antiplasmin and circulating tPA or
urokinase
○​ Clot lysis yields FDPs: interfere with normal platelet aggregation
1.​ E-nodules
2.​ D-dimers - marker of thrombosis or other conditions in which significant
activation of fibrinolytic system is present

Larger fragments may be incorporated in the clot resulting to instability in cases

of severe coagulopathy (hyperfibrinolysis assoc. With trauma-induced
coagulopathy or DIC)

○​ TAFI: removes lysine residues from fibrin which are essential for binding
plasminogen
 CONGENITAL FACTOR DEFICIENCIES
3 most frequent:
1.​ F8 deficiency (Hemophilia A, von Willebrand’s Disease)
2.​ F9 deficiency (Hemophilia B, Christmas Disease)
●​ Severe Hemophilia
○​ Spontaneous bleeds, frequently into joints → crippling arthropathies
○​ Clinical sequelae: Intracranial bleeding, intramuscular hematomas,
retroperitoneal hematomas, and gastrointestinal, genitourinary, and
retropharyngeal bleeding in severe disease
○​ Moderately severe: Less spontaneous bleeding, likely to bleed severely
after trauma or surgery
○​ Mild: Do not bleed spontaneously, have only minor bleeding after major
trauma or surgery
●​ Platelet function: Normal
●​ Diagnosis is not made until after first minor procedure
3.​ F11 deficiency

F8 F9 F11

Inheritance Pattern Sex-linked recessive Sex-linked recessive Autosomal recessive

Incidence Males Males Ashkenazi Jews

Clinical Severity Depends on Depends on Bleeding may occur

measurable level of measurable level of after surgery, trauma,
F8 in patient’s F9 in patient’s or invasive
plasma plasma
procedures
<1% Severe disease

1-5% Moderately severe

5-30% Mild disease

Treatment F8 concentrate F9 concentrate FFP

1mL plasma = 1 unit
of F11 activity

Recombinant F8: Recombinant or Factor 7a: for

patients not treated high-purity F9 patients with
previously, HIV and anti-factor 11
HCV seronegative antibodies

Antifibrinolytics in
women with
menorrhagia

Prevalence 20% develop

 inhibitors that can
neutralize F8

●​ Activity levels should be restored to:

○​ 30-40% - mild hemorrhage
○​ 50% - severe bleeding
○​ 80-100% - life-threatening bleeding

von Willebrand’s Disease

●​ Most common congenital bleeding disorder
●​ Quantitative/qualitative defect in vWF
○​ vWF carries F8
○​ Important for normal platelet adhesion to exposed subendothelium
○​ Important for aggregation under high-shear conditions
●​ Bleeding is characteristic of platelet disorders: easy bruising, mucosal bleeding,
menorrhagia common in women
●​ 3 types:
1.​ Partial quantitative deficiency
2.​ Qualitative defect
3.​ Total deficiency

I II III

Responds well to May respond, depending on Usually unresponsive, may

desmopressin (DDAVP) particular defect require vWF concentrates

Factor XI Deficiency / Hemophilia C

●​ Autosomal recessive
●​ Ashkenazi Jews, but found in all races
●​ Rare spontaneous bleeding
●​ Volume of FFP needed depends on patient’s baseline level, desired level, and plasma
volume
Others
1.​ Deficiency of FII, FV, FX
2.​ FVII deficiency
3.​ FXIII deficiency (Duckert)

F2, 5, 10 F7 F13

Autosomal recessive Autosomal recessive Autosomal recessive

Significant bleeding in Clinical bleeding vary widely, Severe bleeding diasthesis

homozygotes with <1% uncommon unless level is 3% Male to female = 1:1
normal acitvity

Most common bleeding Only associated bleeding in

manifestation: children: inherited deficiency
●​ Easy bruising
●​ Mucosal bleeding Bleeding delayed due to
(epistaxis, oral normal formation of clots but
mucosal) susceptible to fibrinolysis
●​ Postoperative
bleeding (30% of Umbilical stump bleeding:
surgical procedures) characteristic

High risk of intracranial

bleeding

Spontaneous abortion is
usual unless with
replacement therapy

FFP FFP with recombinant factor FFP

●​ 1 unit of activity per 7a: Excellent hemostasis can Cryoprecipitate
mL be achieved with frequent F13 concentrate
●​ Except F5 d/t inherent infusions
instability 1-2% usually adequate for
●​ Daily infusion for F5 hemostasis
for 20-25% activity

Prothrombin complex
concentrates for FX

Half life of FII: 72 hours Half life of recombinant F7a:

~2 hours

Half life of F7 in FFP: 4 hours

FV may be co-inherited with

FVIII
●​ Tx: F8 concentrate +
FFP
 PLATELET FUNCTIONAL DEFECTS

TESTS OF HEMOSTASIS AND BLOOD COAGULATION

1.​ Careful review of patient’s clinical history including previous abnormal bleeding or
bruising, drug use, basic laboratory testing - initial approach to assess hemostatic
function
2.​ Conventional Coagulation Tests - does not reflect complexity of in vivo coagulation,
poorly reflect status of actively bleeding patient as it uses only plasma, not whole blood
a.​ Platelet Count
b.​ PT
c.​ INR
d.​ aPTT
3.​ Viscoelastic Assays - monitor hemostasis as dynamic process, better assess complex
hemostatic function of an actively bleeding patient
a.​ TEG
b.​ ROTEM (Rotational Thromboelastometry)

Conventional Coagulation Tests

Platelet Count

Normal 150 000 - 400 000/uL

Bleeding or thrombotic complications > 1 000 000/uL
Increased bleeding complications with major surgical procedures < 50 000/uL
Increased bleeding complications with minor surgical procedures < 30 000/uL
Spontaneous hemorrhage < 20 000/uL
Still recommended in ophthalmologic and neurosurgical procedures at < 100 000/uL

Prothrombin Time (PT)

●​ Reagent: when added to plasma leads to formation of fibrin clot
1.​ Thromboplastin
2.​ Calcium
●​ Measures extrinsic pathway
●​ Measures Factors I, II, V, VII, X
○​ Factor VII - part of extrinsic pathway
■​ Shortest half-life
■​ Vitamin K-dependent
○​ Factors I, II, V, X - part of common pathway
●​ To detect abnormal coagulation caused by Vitamin K deficiencies and warfarin therapy
●​ Difficult to assess the degree of anticoagulation on basis of PT alone
International Normalized Ratio (INR)
●​ International Sensitivity Index (ISI) - unique to each batch of thromboplastin furnished by
manufacturer
○​ Human brain: ISI 1
○​ Optimal reagent: 1.3 - 1.5
𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑃𝑇 𝐼𝑆𝐼
●​ Formula: 𝐼𝑁𝑅 = ( 𝑛𝑜𝑟𝑚𝑎𝑙 𝑃𝑇
)

Activated Partial Thromboplastin Time

●​ Reagent: in the presence of plasma leads to fibrin clot formation
1.​ Phospholipid substitute
2.​ Activator
3.​ Calcium
●​ Measures function of Factors I, II, V of common pathway
●​ Measures function of Factors VIII, IX, X and XII of intrinsic pathway
●​ Monitors heparin therapy with therapeutic range of 1.5 - 2.5 x control value (approx. 50 -
80 seconds)
○​ Low molecular weight heparins - selective Xa inhibitors
■​ Mildly elevate aPTT
○​ Therapeutic monitoring not routinely recommended

PT aPTT INR
Reagent Thromboplastin Phospholipid
Calcium substitute
Activator
Calcium
Factors measured I, II, V, VII, X I, II, V of common Thromboplastin (III)
VIII, IX, X and XII of
intrinsic
Measurement for Abnormal coagulation Monitors heparin Degree of
caused by Vitamin K therapy with anticoagulation
deficiencies and therapeutic range of
warfarin therapy 1.5 - 2.5 x control
value (approx. 50 - 80
seconds)

Note:
●​ Medications may significantly impair hemostatic function such as:
○​ Antiplatelet agents (clopidogrel, GP IIb/IIIa inhibitors)
 ○​ Anticoagulant agents (hirudin, chondroitin sulfate, dermatan sulfate
○​ Thrombolytic agents (streptokinase, tPA)
●​ If abnormalities in any coagulation studies cannot be explained by any known
medications, congenital abnormalities of coagulation or comorbid disease should be
considered

Viscoelastic Assays
●​ Both TEG and ROTEM measure viscoelastic properties of blood as clotting is induced
under a low shear environment
○​ Shear elasticity determines
■​ Kinetics of clot formation and growth - determine adequacy of quantitative
factors available for clot formation
■​ Strength and stability of formed clot - provides information about ability of
clot to perform work of hemostasis

EVALUATION OF EXCESSIVE INTRAOPERATIVE OR POSTOPERATIVE BLEEDING

May be the result of:
1.​ Ineffective hemostasis - failure to control bleeding
2.​ Blood transfusion (i.e. dilutional coagulopathy - clotting factors are diluted causing
bleeding)
3.​ Undetected hemostatic effect - anticoagulants leads to delayed bleeding
4.​ Consumptive coagulopathy - excessive activation of coagulation system
5.​ Fibrinolysis - uncontrolled bleeding if excessive
6.​ Inadequate mechanical hemostasis - unassociated with bleeding from other sites

Massive blood transfusion Causes thrombocytopenia

Bleeding following massive ●​ Hypothermia - low body temperature impairs

transfusion d/t clotting factors, ineffective platelet aggregation
●​ Dilutional coagulopathy - dilute clotting factors and
platelets causing deficiency of key coagulation
proteins
●​ Platelet dysfunction - reduced ability of platelets to
form an adequate plug
●​ Fibrinolysis - hyperfibrinolysis where breakdown of
fibrin clots is accelerated leading to uncontrolled
bleeding
●​ Hypofibrinogenemia - dilution of fibrinogen thus
diminished ability to form stable clots

Hemolytic transfusion reaction ●​ Diffuse bleeding = first sign of transfusion reaction

●​ Related to release of ADP from hemolyzed RBCs
→ diffuse platelet aggregation
Transfusion purpura Donor platelets are of uncommon HPA-1 group
●​ Uncommon cause of thrombocytopenia and
bleeding after transfusion
○​ Platelets sensitize the recipient
○​ Recipient makes antibody to foreign platelet
antigen
○​ Foreign platelet antigen does not completely
disappear from recipient circulation but
attaches to own platelets
○​ Antibody destroys recipient’s own platelets
○​ Resultant thrombocytopenia and bleeding
may continue for several weeks
●​ C/I: Platelet transfusion d/t binding of antigen and
damage from antibody
●​ Tx: Corticosteroids to reduce bleeding tendency

Posttransfusion purpura ●​ Self-limited

●​ Passage of several weeks leads to subsidence

DIC ●​ Systemic activation of coagulation system

●​ Deposition of fibrin clots and microvascular
ischemia
●​ May contribute to the development of multiorgan
failure
●​ Severe bleeding complications
○​ Consumption
○​ Subsequent exhaustion of coagulation
proteins and platelets d/t ongoing activation
of coagulation system

Severe hemorrhagic disorders Gram-negative sepsis

d/t thrombocytopenia ●​ Defibrination and hemostatic failure may occur with:
○​ Meningococcemia
○​ C. perfringens sepsis
○​ Staphylococcal sepsis
●​ Hemolysis = one mechanism in sepsis leading to
defibrination

LEVELS OF CONCERN