hemostasis & blood clotting

coagulation / clotting

the normal, protective process whereby blood changes from a liquid to solid or semi-solid state in response to vascular injury

• may result in hemostasis

hemostasis

the cessation of blood loss from a vascular injury

thrombosis

a pathologic process in which blood clots form inside blood vessels

fibrinolysis

breakdown of the blood clot

• part of hemostasis

• breaks down the fibrin meshwork

plasma

the liquid portion of the blood

• CAN clot (contains all clotting factors)

serum

the liquid portion left over after blood has clotted

• CANNOT clot

events of blood coagulation (hemostasis)

1. vasoconstriction
2. platelet activation
3. formation of fibrin meshwork
4. fibrinolysis

vasoconstriction

limits blood flow to the site of injury

primary hemostasis

platelet activation

• forms a platelet plug

• adhesion (to subendothelium), aggregation, secretion

secondary hemostasis

formation of the fibrin meshwork by coagulation cascade

• binds blood cells together

• forms hemostatic plug

clot dissolution (fibrinolysis)

removes clots during wound healing

• one step in the thrombolysis

thrombocytes (platelets)

derived from megakaryocytes in bone marrow; small, discoid, enucleated cell fragments

• doesn't divide

• lifespan: 7 - 10 days

• contains glycogen & mitochondria (can perform aerobic metabolism)

open canalicular system

invaginatons of plasma membrane increase surface area available for clotting reactions

alpha granules

contain PROTIENS to be secreted upon activation (e.g. some coagulation proteins like thrombin, fibrinogen, von Willebrand factor)

dense ganules

contain SMALL MOLECULES to be secreted upon activation

• e.g. ADP, serotonin, Ca²⁺

microtubules (actin/myosin network)

maintain discoid shape and mediate morphological changes upon activation

• e.g. extension of pseudopodia

activated platelets

• dramatically change their shape

• change their surface properties (become sticky for adhesion)

• secrete contents of their granules

platelet activation process

1. adhesion

2. aggregation

3. secretion of granule content

adhesion

multiple interactions between platelet membrane glycoproteins (GPs) and components of the denuded subendothelium

initial trigger of activation

• binding of GP1b to collagen via von Willebrand factor (vWF)

• binding of GP1a to collagen directly

Bernard-Soulier syndrome

autosomal recessive deficiency of GP1b (defective primary hemostasis)

• macrothrombocytopenia, easy bruising, heavy and prolonged bleeding

• affects platelet adhesion

platelet aggregation

activated platelets adhere to other activated platelets

mediated by:

• adhesive receptors GPIIb/GPIIIa (integrins) on the platelet membrane that bind to fibrinogen

• fibrinogen forms a bridge between activated platelets

Glanzmann's thrombasthenia

autosomal recessive deficiency of GPIIb/GPIIIa (defective primary hemostasis)

• easy bruising, heavy prolonged bleeding

• affects platelet aggregation

Plavix (clopidogrel)

antithrombotic drug that inhibits platelet aggregation by interfering with GPIIb/GPIIIa

secretion

upon activation, platelets release a number of substances stored in granules

• dense bodies: ADP, serotonin, Ca²⁺

• alpha granules: vWF, fibrinogen, coagulation cascade proteins

ADP, serotonin, Ca²⁺

secreted from dense bodies upon platelet activation

adenosine diphosphate (ADP)

secreted from dense bodies: platelet activator

serotonin

secreted from dense bodies: vasoconstrictor

calcium (Ca²⁺)

secreted from dense bodies: activator of coagulation cascade (necessary for secondary hemostasis)

• acts as signal to other platelets to activate eicosanoid production

vWF, fibrinogen, coagulation cascade proteins

secreted from alpha granules upon platelet activation

fibrinogen

promotes platelet aggregation & converted to fibrin to form mesh

coagulation cascade proteins

i.e. prothrombin & thrombin (acts as activator of platelets + key role in coagulation cascade)

• most other clotting proteins are produced by the liver and released into blood

von Willebrand factor (vWF)

a large multimeric glycoprotein (polymer up to 80 polypeptides) synthesized only in endothelial cells & megakaryocytes

• found in subendothelial matrix, in circulation, & in platelet alpha granules

• promotes adhesion of platelets to subendothelium to initiate activation

• stabilizes clotting factor VIII in circulation (forms complex with Factor VIII in circulation)

von Willebrand disease (vWD)

caused by mutations in vWF gene encoding vWF

~ 1 in 100 people have defect in vWF (majority subclinical)
~ 1 in 8000 have clinical diagnosis (more common than hemophilia A)

• most common congenital bleeding disorder

• autosome dominant or recessive: allelic heterogeneity

symptoms:

• considerable clinical heterogeneity due to allelic heterogeneity

  • 3 clinical types with subtypes

• bleeding diathesis due to defective platelet adhesion

  • easy & excessive bleeding following injury, easy bruising, heavy or prolonged menstruation

• in severe cases, there is a secondary Factor VIII deficiency

  • can lead to hemophilia A misdiagnosis

thromboxanes

a member of eicosanoid family of lipids

following platelet activation

1. phospholipase A₂ (PLA₂) is activated

2. PLA₂ cleaves arachidonic acid from membrane phospholipids

3. arachidonic acid is converted to intermediary prostaglandins by cyclooxygenase (COX) enzymes

4. prostaglandins are → — A₂

thromboxane A₂

a potent activator of platelets and promotes vasoconstriction

aspirin

nonsteroidal anti-inflammatory (NSAID)

• (suicide inhibitor) of COX1 & COX2

• platelet inhibitor (antithrombotic = inhibits production of thromboxane A₂)

collagen, vWF

platelet activating signals: subendothelial molecules exposed to endothelial damage

platelet activating signals (via positive feedback)

• ADP: activator signal

• fibrinogen: bridge between GPIIb/GPIIIa mediating platelet aggregation

• vWF: needed for adhesion to subendothelium

• thromboxane A₂: activator

• thrombin: activator

• platelet activating factor (PAF): a phospholipid hormone

phosphatidylserine

membrane phospholipid

• exposed to outer leaflet of activated platelet (similar to apoptosis)

• provides a surface for clotting cascade: acts as cofactor

extrinsic pathway

activated by external stimuli (e.g. blunt trauma)

injury → VII + TF → VII to VII_a → TF:VII_a→ TF:VII_a + X → X_a

the BLUE pathway

intrinsic pathway

activated by internal stimuli (e.g. clotting in a test tube)

surface contact → XII to XII_a → XII_a + XI→ XI_a + IX → IX_a + X→ X_a

the RED pathway

Factor X

initiates the common pathway

• convergence of the intrinsic & extrinsic pathway

• activation of thrombin & production of fibrin

zymogen

most clotting factors are synthesized in the liver & secreted into the plasma in their — (precursor) form

• available to be quickly activated

phospholipid (PL), calcium (Ca²⁺)

most steps in the coagulation cascade happen on a membrane and require — & —

clotting reaction activation

1. a SERINE protease binds to its protein cofactor on a membrane surface

2. protease cuts a peptide bond in zymogen (substrate) releasing the active form

3. products catalyze the next proteolytic step in the cascade

4. cofactors accelerate clotting 10000-fold

common pathway

the GREEN pathway

tissue factor (factor III)

exposed upon damage to endothelium; required for initiation of the extrinsic pathway in response to vascular trauma

• a cell-surface protein found in cells in subendothelium (similar to vWF)

• cofactor for FACTOR VIIa

factor VIIa

acts on the membrane surface & requires the following cofactors:

• phospholipid (PL)

• Ca²⁺

• TF (required for activity)

activates X (extrinsic pathway) & IX (intrinsic pathway)

factor VII

binds to tissue factor & autocatalyzes its activation to VIIa

fibrinogen

composed of 1 triple-helices of α, β, & γ chains joined at N-termini = 6 chains total per molecule

N-terminal ends are highly negatively charged (prevents aggregation by repulsion)

thrombin

cleaves the N-termini of fibrinogen

soft clot

aggregation of of fibrin

hard clot

covalent cross-linking by Factor XIIIa stabilizes the firbin clot

factor XIIIa

transglutaminase = catalyzes a transamination between gln & lys in neighboring fibrin monomers (hard clot)

• only enzyme of the clotting cascade that is NOT a serine protease

• creates a strong 3-D network resistant to mechanical and proteolytic damage

• traps aggregating platelets forming the clot (hemostatic plug)

bleeding time

evaluation of platelet function

• standardized small incisions are made in the arm

• time for bleeding to stop is measured

• poor reproducibility and invasive; largely replaced

platelet aggregometry

in vitro evaluation of platelet function

• platelets are isolated in plasma and activated with ADP (or another platelet activator)

• time for platelets to aggregate is measured (normal range varies by lab)

prothrombin time (PT)

evaluation of EXTRINSIC clotting cascade activity (including common pathway)

• clotting of plasma is induced by addition of tissue factor (TF)

• clotting factors required: I (fibrinogen), II (prothrombin), V, VII, X

• time for plasma to clot is measured (normal = 10 - 13 secs)

partial thromboplastin time (PTT)

evaluation of INTRINSIC clotting cascade activity (including common pathway)

• clotting of plasma is induced by addition of silica (or other activator of intrinsic pathway)

• clotting factors required: I (fibrinogen), II (prothrombin), V, VIII, IX, X, XI, XII

• time for plasma to clot is measured (normal = 30 - 50 secs)

hemophilia A

gene: F8 (due to unequal crossing over between inverted repeats which results in an inversion = null mutation)

protein: factor VIII

incidence: 1 in 4000 male births

hemophilia B

also called Christmas disease after the patient

gene: F9 (due to unequal crossing over between inverted repeats which results in an inversion = null mutation)

protein: factor IX

incidence: 1 in 20000 male births

hemophilia A/B

X-linked bleeding disorder caused by the deficiency of clotting factors

symptoms:

• spontaneous bleeding in joints and skeletal muscles

• arthritis

• prolonged bleeding

labs:

• bleeding test: normal

• PTT: prolonged

• PT: normal

treatment:

• recombinant clotting factors now available; administered by IV

anticoagulant

proteins which prevent clotting beyond the injured area

antithrombin

serpin (serine protease inhibitor) = irreversibly inactivates serine proteases

• one of the most important anticoagulant

• protein-inhibitor complexes are removed from the circulation by the liver

factor Xa, thrombin

the main targets of antithrombin is — & —

heparin

a glycosaminoglycan (GAG) that is used as an anticoagulant

• stimulated antithrombin

  • 17000-fold antithrombin activation for Xa substrate

  • 9000-fold antithrombin activation for thrombin substrate

• injected therefore accelerates inactivation of Xa & thrombin

• treatment for venous thrombosis

fondaparinux (F)

a pentasaccharide related to Low-Molecular Weight Heparin (LMWH) & more specific than High-Molecular Weight Heparin (HMWH)

• specific inhibitor of Xa

antithrombogenic

endothelial cell surfaces are —

thrombomodulin

located on endothelial cell surface that binds to thrombin

• thrombin substrate specificity changes

• activation of PROTEIN C

protein C

activated by thrombomodulin from endothelial cells

activated protein C (APC)

forms a complex with PROTEIN S

• proteolytically destroy Factors Va & VIIIa

• inhibits the clotting cascade

protein S

forms a complex with activated protein C (APC)

• not a serine protease

factor V Leiden

most common cause of hypercoagulability in Caucasians

• autosomal semi-dominant

• a point mutation in factor V near the APC cleavage site

• resistant to proteolysis by activated protein C (APC) = more likely to stay active in endothelial cell surfaces

heterozygotes have 6 - 8 fold increased risk for deep-vein thrombosis

homozygotes have 30 - 60 fold increased risk for deep-vein thrombosis

plasmin

a serine protease that degrade fibrin clots

• circulates in the plasma as an inactive zymogen

• free — is immediately inactivated by anti- —

plasminogen

zymogen of plasmin

- activated by tissue-type — activator (tPA) & urinary-type — activator (uPA; urokinase)

antithrombogenic agents

• prostacyclin (PLI₂) + nitric oxide (NO)- inhibits adhesion and aggregation

• thrombomodulin & heparan sulfate (heparin-like)

• tissue factor inhibitor

• plasminogen activator (tPA)

vitamin K dependent carboxylation

γ carboxylation of glutamate residues

• carboxyl groups chelate Ca²⁺ which forms a bridge to membrane phospholipids (necessary for localization of clotting factors @ cell surface )

vitamin K

is oxidized in the carboxylase reaction

must be recycled by a 2-step reduction

factor II (prothrombin), VII, IX, X, protein C, protein S

factors that undergo vitamin K-dependent gamma carboxylation of glutamate residues (via vitamin K dependent carboxylase)

coumarin (warfarin)

antagonize vitamin K-dependent gamma carboxylation of several clotting factors = competitive inhibitor

• has to be controlled so that only partial inhibition of the carboxylation is achieved to prevent bleeding complications

• action can be reversed by administration of vitamin K

• being replaced by direct inhibitors

plasminogen activator (PA)

used to treat myocardial infarction & stroke

• promote dissolution of thrombi by locally activating plasminogen to plasmin

• indications: myocardial infarctions, ischemic stroke, pulmonary embolism (only in severe cases)

Activase

thrombolytic- tissue-type plasminogen activator (tPA)

• action cannot be reversed by administration of vitamin K

Abbokinase

thrombolytic- urinary-type plasminogen activator (uPA; urokinase)

• action cannot be reversed by administration of vitamin K

Streptase

thrombolytic- streptokinase

• action cannot be reversed by administration of vitamin K