L-13 Hemostasis I

• Lecture covers Hemostasis I

Learning Objectives

• Describe platelets, their composition, and activation.
• List the four phases of hemostasis.
• Describe blood coagulation.

Hemostasis

• Processes that stop bleeding and prevent blood from leaving a damaged blood vessel.
• Damage to vessel walls is repaired by hemostasis. (hemo = blood, stasis = stop)
• The human hemostatic system provides a natural balance between procoagulant and anticoagulant forces.
• Integrity of blood vessels is needed to maintain proper blood circulation.
• If blood loss goes unchecked, circulating blood volume decreases, this compromises organ/tissue perfusion.

Stages of hemostasis

• Vasoconstriction: The blood vessel narrows
• Platelet plug formation: Platelets form a plug to seal the damaged area
• Coagulation cascade: A series of reactions that activate clotting factors
• Fibrinolysis: The clot is broken down
• Primary Hemostasis: forms a weak platelet plug and is mediated by interactions between platelets and the vessel wall
• Secondary Hemostasis: stabilizes the platelet plug and is mediated by the coagulation cascade

The major components of the hemostatic system:

• Platelets and other formed elements, such as monocytes and red blood cells
• Plasma proteins (the coagulation and fibrinolytic factors and inhibitors)
• The vessel wall
• Platelets ensure that no blood is lost at capillary/venule junctions during normal physiological conditions.
• Failure to prevent blood leakage at the capillary/ venule junction can be seen in case of exercise-induced vasculitis (hiker's rash) after prolonged hikes and/or during strenuous physical exercise – especially in hot weather.

Describe platelets, their composition and activation

• Without hemostasis, even a small injury would be life-threatening.
• Hemostasis involves a balance between coagulant and anticoagulant forces. - Procoagulant forces lead to platelet adhesion, aggregation, and fibrin clot formation. - Anticoagulant forces lead to natural inhibitors of coagulation and fibrinolysis.
• Under normal conditions, hemostasis promotes blood flow.
• Platelets (aka thrombocytes) lack nuclei.
• Platelets are granular cells derived from megakaryocytes.
• Platelets have a lifespan of 7– 10 days.
• They circulate freely and kept mobile but inactive by molecules, secreted by endothelial cells lining the blood vessels, like nitric oxide and prostacyclin.

Platelets

• Platelets are essential for the clotting process that occurs in plasma when blood vessels are ruptured, or their lining is injured.
• By sticking to the damaged site, platelets form a temporary plug that helps seal the break.
• They are the primary cells responsible for hemostasis.
• Platelets contribute their hemostatic capacity via adhesion, activation and aggregation, which are triggered upon tissue injury.
• They stimulate the coagulation factors and other mediators to achieve hemostasis.
• Platelets are the smallest blood component (2–4 µm).
• Normal platelet counts are between 150K and 400K per microliter of blood.
• At any one time, up to a third of circulating platelets are sequestered within the spleen.

Platelets Structure

• Platelets exhibit a biconvex discoid shape, which is maintained by a circumferential bundle of microtubules.
• There are two tubule systems: the dense tubular system and the surface-opening canalicular system.
  • Open canalicular system: invaginations that provide space for platelet products to enter/exit.
• Glycogen granules provide energy source.
• Alpha granules contain fibrinogen, thrombospodin, Factor V, von Willebrand factor (vWF), beta-thromboglobuline (β-TG) and Factor IV.
• Dense granules store non-metabolic ATP, ADP, serotonin and calcium, preventing cell stickiness.
  • Upon activation, platelets release their granules.
• Lambda granules are lysosomes
• Platelets don't have nuclei or organelles other than mitochondria.

Formation of platelets

• A hormone called thrombopoietin regulates the formation of platelets.
• The hematopoietic stem cell produces megakaryocytes.
• The plasma membrane of the megakaryocyte fragments, liberating the platelets.

The four phases of hemostasis

• The hemostasis response is fast, localized, and carefully controlled.
• It involves many clotting factors normally present in plasma as well as several substances that are released by platelets and injured tissue cells.
• During hemostasis, three steps occur in rapid sequence:
  • vascular spasm,
  • Platelet plug formation,
  • coagulation (blood clotting).
• Following hemostasis, the clot retracts.
• It dissolves as it is replaced by fibrous tissue that permanently prevents blood loss.

List the four phases of hemostasis

• Vasoconstriction (vascular spasms): Can significantly reduce blood loss for 20–30 minutes, allowing time for the next two steps to occur.
• Temporary platelet plug -> Primary hemostasis
  • Forms a framework for the developing clot and temporarily seals the vessel wall, minimizing blood loss.
  • This step can be measured clinically as “bleeding time.”
• Coagulation -> Secondary hemostasis
  • Circulating coagulation factors undergo a cascade of reactions that ultimately generate fibrin.
  • Fibrin forms a mesh that stabilizes platelets in the clot.
  • The clot remains until the underlying damage gets repaired.
• Fibrinolysis -> restores function
  • Dismantling of the fibrin clot by specific enzymatic factors and restoration of normal blood flow.

Describe blood coagulation

• First, vasoconstriction Occurs. When endothelial injury happens, nerves connected to the endothelial and smooth muscles cells undergo a reflexive contraction (vascular spasm). It decreases blood flow and minimize blood loss
• Normally (in absence of injury), endothelial cells secrete nitric oxide (NO) and prostaglandins (vasodilation).
• When injury occurs, endothelial cells decrease the production of vasodilators

Primary Hemostasis: Temporary platelet plug

• Intact endothelial cells release nitric oxide and a prostaglandin → prevent platelets from sticking to each other or to blood vessels.
• In response to injury, the underlying collagen fibers are exposed.
  • Adhesion: platelets adhere to the collagen fibers.
  • Activation: platelets swell, form spiked processes, and become stickier.
  • Aggregation: positive feedback loop that augments (adds/increases) the plug.
• Activated platelets produce: ADP, Serotonin and thromboxane A2
  • ADP: a potent aggregating agent that causes more platelets to stick to the area and release granules contents.
  • Serotonin and thromboxane A2: messengers that enhance vascular spasm and platelet aggregation.

Temporary platelet plug - Primary Homeostasis

• Exposure when damaged endothelial cells expose the collagen at injury site.
• Disc-shaped inactive platelets with granules are pulled to the edges of the vessel.
• Endothelial cells release von Willebrand's factor (vWF) that binds to collagen
  • which is a protein bridge, linking platelets to the exposed collagen in the injured vessel wall.
• Platelets can bind directly to collagen via α2β1 integrins.
• Platelet receptor glycoprotein 1b (Gp1b) binds to vWF.
• Gp1b receptor on platelets also bind to other platelet receptors (Gpla, Gplla and GpVI) bind directly to the subendothelial collagen.
• This leads to cytoskeletal alterations that make the platelet "flat” and lead to the formation of podia "tube feet".

Temporary platelet plug - Primary Hemostasis Activation

• When platelets bind to von Willebrand's factor (vWF), they get activated
• Platelets change their shape from disc-shaped structures to "spiky" structures with filopodia; this morphological change increases platelet surface area.
• Secrete more vWF, and serotonin, which attracts more platelets
• Secrete calcium, which will be useful in secondary hemostasis!
• Secrete Thrombin, ADP, and thromboxane A2, which promote additional platelet recruitment and activation.
• The "release reaction" occurs within 2 minutes of adhesion
  • Release of bioactive contents is triggered by intracellular [Ca2+] influx
  • The Delta (dense) granules contain Ca2+, ATP, serotonin, histamine and adrenaline
  • Alpha granules contain fibrinogen, von Willebrand factor, factors V, XI and XIII
  • Platelets also synthesize thromboxane A2 (TXA2) from arachidonic acid. TXA2 is a potent vasoconstrictor
  • When platelets are expressing ADP and TXA2, they up-regulate (change conformation of) another surface receptor, the GPIIB/IIIA. By then, those platelets are fully activated.
• Platelets have a secondary role:
  • Tissue repair: platelets stimulate wound healing by secretion of platelet-derived growth factor, which stimulate mitogenesis of vascular smooth muscle cells and fibroblasts. It promotes cell division and growth!

Temporary platelet plug - Primary Hemostasis Aggregation

• ADP and Thromboxane A2 cause platelets to bind collagen.
• GPIIB/IIIA receptors bind fibrinogen and clumps platelets into the plug.
• As more platelets get activated, they further aggregate, causing a positive feedback cycle that builds a platelet plug within a minute.
• The platelet plug is sufficient to seal “wear and tear" of small blood vessels.
• Because platelet plugs are loosely knit, larger breaks need additional reinforcement.
  • Hence, Secondary Hemostasis!

Coagulation

• Reinforces the platelet plug with fibrin threads that act as a “molecular glue" for the aggregated platelets
• Blood is transformed to a gel in a multistep process that involves a series of clotting factors, or procoagulants
• All procoagulants (except tissue factor) normally circulate in blood in inactive form until needed
• Prostaglandins and NO, which are secreted by undamaged cells and prevent platelets activation
• Inappropriate hemostasis impairs tissue perfusion and result in ischemia.

Coagulation - Bleeding disorders

• Bleeding disorders can occur when there are issues with the process of hemostasis.
• Symptoms of hemophilia include: nosebleeds that are hard to stop, gums that bleed when brushing, or wounds that take a long time to heal. Symptoms vary depending on the severity of the disease.
• Treatments include blood thinners, surgery, or clot-dissolving medications.

Summary of primary hemostasis

• When damage to endothelial cells takes place, there's local up-regulation of a ligand called von Willebrand's factor from the type-4 sub-endothelial collagen (at the basement membrane).
• Exposed collagen binds circulating von Willebrand's factor (vWF), which then binds Glycoprotein 1b receptors, Gp-1b on platelets.
• The platelets also have Gp-2b/IIIa receptors (activated via ADP), which interact with fibrinogen (TXA2), allowing platelet aggregation and formation of a temporary hemostatic plug.
• Notice that at this stage the fibrinogen is in precursor form (not yet cleaved to fibrin).
• Primary Hemostasis For the next stage of coagulation, the enzyme thrombin cleaves fibrinogen into fibrin, forming a more stable platelet plug.

Primary hemostasis summary

• Clotting factors get proteolytically activated to:
  • activate fibrin, or Factor IA.
  • form a fibrin mesh (the cement)
• Factor X gets activated to activate a common coagulation cascade - next lecture

Anti-platelet drugs

• Medications that prevent blood clots by making blood less sticky.
• Often prescribed to people who have had a heart attack, stroke, or stent placement.
• Examples:
  • Aspirin, the first antiplatelet drug and is a cyclooxygenase inhibitor
  • Clopidogrel (Plavix), Prasugrel (Effient), Ticagrelor (Brilinta) are all ADP receptor inhibitor that makes platelets less sticky
  • Dipyridamole is an adenosine reuptake inhibitor that blocks enzymes involved in clotting
  • Cilostazol is a phosphodiesterase inhibitor that widens blood vessels and stops platelets from sticking together.