4. hemostasis

Hemostasis

“arrest of bleeding”

Process of blood clotting, followed by dissolution of the clot once the injured
tissue is repaired

4 coordinated events in response to loss of vascular integrity (steps of hemostasis)

1. Vascular constriction

2. Platelet activation, adhesion, and aggregation → temporary, loose platelet plug

3. Coagulation → fibrin synthesis and formation of thrombus (clot)

4. Dissolution of the clot following tissue repair

Endothelial cells normally inhibit clot formation by Endothelial secretion of:

Prostacyclin (PGI2) + Nitric oxide (NO)

Prostacyclin (PGI2)

inhibitor of platelet function

Nitric oxide (NO)

vasodilator and inhibitor of platelet activation and aggregation

Endothelial cells normally inhibit clot formation by Expression of:

Heparin sulfate (HS)

Thrombomodulin (TM)

Tissue factor pathway inhibitor (TFPI)

Heparin sulfate (HS)

activates antithrombin III

Thrombomodulin (TM)

changes the affinity of thrombin from procoagulation factors toward anticoagulation factors

Tissue factor pathway inhibitor (TFPI) 

inhibits TF-VIIa/Xa complex

primary vs secondary hemostasis

primary hemostasis: Vascular constriction

Immediate reduced blood flow to the injured area

Mediated by the local secretion of endothelin

Transient effect → bleeding resumes if clot is not formed

endothelin

potent endothelium-derived vasoconstrictor

Megakaryocytes

large cells produced in the bone marrow by endomitosis → single voluminous, polyploid cells

precursor of platelets

endomitosis

nuclear division without cytoplasmic division

how many platelets bud off from a single mature megakaryocytes + what stimulates this process

Approx. 4000

Stimulated by thrombopoietin

Platelets (thrombocytes) + life span

anuclear, lack most organelles, but have a complex cytoskeleton to maintain shape 

Lifespan ~ 10 days

types of platelet cytoplasmic granules

dense

alpha

lysosomal

Dense platelet cytoplasmic graules

Ca2+, ATP/ADP, serotonin

Alpha platelet cytoplasmic granules contain 

fibrinogen, von Willebrand factor (vWF), factor V

Lysosomal platelet cytoplasmic granules contain

hydrolytic enzymes

platelet membrane glycoprotein receptors:

GPIb

GPIa/IIa

GPIIb/IIIa

platelet membrane glycoprotein receptors: GPIb

binds to vWF immobilized on subendothelial collagen

platelet membrane glycoprotein receptors:  GPIa/IIa

binds to collagen

platelet membrane glycoprotein receptors: GPIIb/IIIa

binds to free fibrinogen and vWF

von Willebrand factor

Large glycoprotein secreted by platelets (a-granules) and endothelial cells

Heterogeneous multimers linked by disulfide bonds

when are von Willebrand factors released + what occurs 

Released from endothelial cells in response to damage

Forms a bridge between a glycoprotein complex on the surface of
platelets and exposed collagen fibrils

what do von Willebrand factors have binding sites for

Collagen

GPIb, GPIIb/IIIa

Factor VIII → transport and survival

Inherited deficiencies: von Willebrand disease

Most common inherited bleeding disorder (patient history before tooth extraction!!!)

Affects both platelet adherence and coagulation cascade (FVIII)

primary hemostasis: platelet actiation overview steps

1. platelets triggered

2. adhesion

3. secretion

4. shape change

5. aggregation

6. platelet plug formation

1. Platelet activation Triggers

Exposed subendothelial collagen

ADP – from platelet degranulation

TXA2 – secreted by activated platelets

Thrombin – from coagulation cascade

2. Adhesion

refers to the platelet-subendothelial interaction at the site of injury

GPIb binds to vWF immobilized on exposed collagen

GPIa/IIa bind to directly to collagen → conformational change to expose GPIIb/IIIa to bind more vWF and free fibrinogen

3. Secretion

degranulation of dense and alpha-granules

Release of ADP (potent platelet activator), vWF, serotonin (vasoconstrictor), Ca2+

4. Shape change

from discoidal to irregular with long pseudopods

Increased surface → increased ability to bind to other platelets

Expression of phosphatidylserine (PS)

5. Aggregation

recruitment of other platelets

Exposed GPIIb/IIIa bind to fibrinogen and vWF to form bridges with neighboring platelets

6. Platelet plug formation

3-5min

1. Platelet adhesion to exposed subendothelial collagen directly (GPIa/IIa) or mediated by vWF (bridge btw collagen and GPIb)

2. Adhesion followed by shape change and release of various procoagulants (ADP, TXA2, fibrinogen)

3. Secreted procoagulants aid in recruitment of additional platelets and promote their aggregation → primary loose hemostatic plug

secondary hemostasis: Coagulation

Series of amplifying enzymatic reactions that lead to the formation of an insoluble fibrin clot

coagulation factors

Major components are glycoproteins secreted primarily by the liver (except TF and Ca2+)

Most factors are synthesized in zymogen form and become active by proteolysis

Non-proteolytic factors (accessory proteins) are activated by conformational change

zymogen

inactive form that becomes active by proteolysis

accessory proteins are activated by 

conformational change

Complex assembly

Negatively charged phospholipid phosphatidylserine (PS) on activated platelets and injured endothelial membrane

Ca2+ binds to g-carboxylated glutamate residues in factors II,
VII, IX, and X

g-carboxylation of glutamic acid

posttranslational modification of 9-12 glutamate residues (N-term)

Vit. K coenzyme needed

hydroquinone (functional vit K) is oxidized to vit K epoxide, which is reduced to regenerate hydroquinone by Vit K epoxide reductase (VKOR)

Warfarin

anticoagulant

synthetic Vitamin K analog inhibits VKOR 

Vitamin K 

dietary fat-soluble

2 forms: Phyloquinone (K1) + Menaquinone (K2)

Vitamin K: Phyloquinone (K1) 

in green vegetables

Vitamin K: Menaquinone (K2)

produced from K1 by gut bacteria

Coagulation cascade

1. initiation

2. amplification

3. propagation

Coagulation cascade: 1. initiation

extrinsic/ TF-pathway

Tissue factor is available when injury occurs

1. binds to circulating FVII, which is activated (TF-FVIIa)

2. TF-FVIIa activates FX to FXa

3. Initiation of signal by injury, but insufficient Xa production

Where is tissue factor (TF) produced?

in vascular adventitia

Coagulation cascade: 2. amplification

intrinsic/ contact activation pathway

1. Thrombin activates FV, FVII, FVIII, FXI, FXII

2. FIXa complexes with FVIIIa (bound to vWF to increase its half-life) to
   activate FX

3. FXa triggers an explosion of thrombin generation (4000x amplification of signal)

Coagulation cascade: 3. propagation

common pathway

1. FXa activates prothrombin to thrombin (FIIa)

2.  Thrombin converts fibrinogen to fibrin

Fibrinogen

3 pairs of polypeptides (Aa, Bb, g2) covalently linked at the N-termini by disulfide bonds

how is fibrin formed

Thrombin cleaves fibrinopeptides A and B from fibrongen → monomers of
abg2 (fibrin)

weak fibrin mesh

Fibrin spontaneously aggregates in a regular array

insoluble fibrin clot formation

Thrombin activates FXIII to FXIIIa

FXIIIa = transglutaminase that forms cross-links between fibrin monomers → insoluble fibrin clot

overview of hemostasis

1. Release of clotting factors from damaged endothelial cells and activated platelets at the injury site → temporary platelet plug

2. Cascade of chemical reactions → formation of thrombin

3. Formation of fibrin and trapping of blood cells → insoluble blood clot

Clot propagation termination 2 mechanisms:

Antithrombin (antithrombin III)

Protein C pathway

Clot propagation termination mechanisms: Antithrombin (antithrombin III)

Serine protease inhibitor (Serpin) which inactivates thrombin, VIIa, IXa, Xa, and XIa

Activated by heparin

Clot propagation termination mechanisms: Protein C pathway

Endothelial thrombomodulin induces conformational change in thrombin

Altered thrombin is incapable of activating platelets or converting fibrinogen to fibrin, but can activate protein C

Activated protein C + cofactor protein S inactivates Va and VIIIa

Fibrinolysis

process of clot dissolution

Plasminogen is activated by conversion to plasmin by t-PA (tissue plasminogen activator) secreted from vascular endothelial cells and u-PA (urokinase) secreted by a variety of cells

Plasmin cleaves fibrin (and other clotting factors) at multiple sites, releasing fibrin fragments (D-dimers)

Inhibitors of fibrinolysis include:

Plasminogen activator inhibitor 1 (PAI-1)–inhibits t-PA

α2-Antiplasmin (α2AP) = plasma protein -rapidly inhibits free plasmin