Hemostasis I: COM 5855

• This lecture is about Hemostasis I, part of the Hematopoietic and Lymphoreticular System course COM 5855 at Nova Southeastern University.
• The lecture date is scheduled for 03/11/2025.

Learning Objectives

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

Hemostasis Overview

• Hemostasis stops bleeding and prevents blood from leaving damaged blood vessels.
• Hemostasis repairs damage to vessel walls (hemo = blood, stasis = stop).
• The human hemostatic system balances procoagulant and anticoagulant forces.
• Blood vessel integrity is needed for proper blood circulation.
• Unchecked blood loss reduces circulating blood volume, compromising organ/tissue perfusion.

Stages of Hemostasis

• Vasoconstriction narrows the blood vessel.
• Platelet plug formation involves platelets forming a plug to seal the damaged area.
• The coagulation cascade is a series of reactions that activate clotting factors.
• Fibrinolysis breaks down the clot.
• Primary Hemostasis forms a weak platelet plug, mediated by interactions between platelets and the vessel wall.
• Secondary Hemostasis stabilizes the platelet plug and is mediated by the coagulation cascade.

Components of the Hemostatic System

• Platelets and other formed elements like monocytes and red blood cells.
• Plasma proteins which include coagulation and fibrinolytic factors and inhibitors.
• The vessel wall itself is a component.

Platelet Function

• During normal conditions, platelets prevent blood loss at capillary/venule junctions.
• Exercise-induced vasculitis (hiker’s rash) after prolonged hikes or strenuous exercise, especially in hot weather can cause blood leakage at the capillary/venule junction.

Platelets, Composition, and Activation

• Without hemostasis, even a small injury can be life-threatening.
• Hemostasis balances 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 (thrombocytes) lack nuclei.
• Platelets are granular cells derived from megakaryocytes.
• Platelets have a lifespan of 7–10 days.
• Endothelial cells lining blood vessels secrete molecules (nitric oxide, prostacyclin) that keep circulating platelets inactive.

Platelets

• Necessary for the clotting process in plasma when blood vessels rupture or their lining is injured.
• Platelets form a temporary plug to help seal the break by sticking to the damaged site.
• Primary cells responsible for hemostasis.
• Contribute to hemostatic capacity via adhesion, activation, and aggregation
  • Triggered upon tissue injury.
• They stimulate coagulation factors and other mediators to achieve hemostasis.
• Platelets are the smallest blood component, measuring 2–4 µm.
• Normal platelet counts range between 150,000 to 400,000 per microliter of blood.
• Up to a third of circulating platelets are sequestered within the spleen at any given time.

Platelet Structure

• Platelets exhibit a biconvex discoid shape maintained by a circumferential bundle of microtubules.
• There are two tubule systems:
  • The dense tubular system.
  • The surface-opening canalicular system.
• The open canalicular system consists of invaginations that allow space for platelet products to enter/exit.
• Glycogen granules provide energy
• Alpha granules contain fibrinogen (Fib), 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 these granules.
• Lambda granules contain lysosomes.
• Platelets do not have nuclei or organelles other than mitochondria.

Formation of Platelets

• Thrombopoietin regulates the formation of platelets.
• The hematopoietic stem cell produces megakaryocytes.
• The plasma membrane of the megakaryocyte fragments, liberating platelets.

Four Phases of Hemostasis

• The hemostasis response is fast, localized, and controlled.
• It involves many clotting factors normally present in plasma, as well as substances released by platelets and injured tissue cells.
• Three steps occur in rapid sequence:
  • Vascular spasm
  • Platelet plug formation
  • Coagulation (blood clotting).
• Following hemostasis:
  • The clot retracts, and is replaced by fibrous tissue which permanently prevents blood loss

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.
• Can be measured clinically as "bleeding time."

Coagulation -> Secondary Hemostasis

• Circulating coagulation factors undergo a cascade of reactions that generate fibrin.
• Fibrin forms a mesh that stabilizes platelets in the clot.
• The clot remains until the underlying damage is repaired.

Fibrinolysis -> Restores Function

• Dismantling of the fibrin clot by specific enzymatic factors and restoration of normal blood flow.

Vasoconstriction in Blood Coagulation

• When endothelial injury happens, nerve’s connected to the endothelial and smooth muscle cells undergo a reflexive contraction, also known as a vascular spasm
  • This decreases blood flow and minimizes blood loss.
• Normally, endothelial cells secrete nitric oxide (NO) and prostaglandins (vasodilation) in the absence of injury.
• When injury occurs, endothelial cells decrease the production of vasodilators.

Temporary Platelet Plug in Blood Coagulation

• Intact endothelial cells release nitric oxide and prostaglandin (prostacyclin, PGI2) to prevent platelets from sticking to each other or to blood vessels.
• In response to injury:
  • Exposure occurs, which exposes the underlying collagen fibers.
  • Adhesion of Platelets adheres to the collagen fibers.
  • Activation causes platelets to swell, form spiked processes, and become stickier.
  • Aggregation is a positive feedback loop that augments (adds/increases) the plug.
• Activated platelets produce:
  • ADP: A potent aggregating agent that causes more platelets to stick to the area and release granule contents.
  • Serotonin and thromboxane A2: messengers that enhance vascular spasm and platelet aggregation.

Primary Hemostasis: Temporary Platelet Plug and Exposure

• Damaged endothelial cells expose the collagen at the 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.
  • This results in a protein bridge, linking platelets to the exposed collagen in the injured vessel wall.
• Platelets can bind directly to collagen via α2β1 integrins.

Factors in temporary platelet plug

• The platelet receptor glycoprotein 1b (Gp1b) binds to vWF (Adhesion).
• Gp1b receptor on platelets also binds to other platelet receptors (GpIa, GpIIa and GpVI).
  • They bind directly to the subendothelial collagen.
• Alterations to make the platelet "flat” lead to the formation of podia "tube feet".

Activation of the Temporary Platelet Plug

• When platelets bind to von Willebrand's factor (vWF), they get activated.
• Platelets change shape from disc-shaped structures to "spiky" structures with filopodia.
  • This morphological change increases platelet surface area.
• Platelets 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.

Primary Hemostasis: Activation and Platelet Function

• 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.
• Platelets expressing ADP and TXA2, will 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.
  • Promotes cell division and growth.

Primary Hemostasis: Aggregation

• ADP and Thromboxane A2 cause platelets to bind collagen.
  • GPIIB/IIIA receptors bind fibrinogen and clumps platelets into the plug.
• Activated platelets 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.
• Larger breaks need additional reinforcement.
  • This means Secondary Hemostasis!

Secondary Hemostasis in Blood Coagulation

• Reinforces the platelet plug with fibrin threads that act as a “molecular glue" for the aggregated platelets.
• Blood transforms to a gel in a multistep process involving clotting factors or procoagulants.
• All procoagulants (except tissue factor) circulate in blood in an inactive form until needed.

Prevention of Inappropriate Primary Hemostasis

• Undamaged cells secrete prostaglandins and NO, which prevent platelet activation.
• Inappropriate hemostasis impairs tissue perfusion and results in ischemia.

Coagulation and Bleeding Disorders

• Bleeding disorders occur when there are hemostasis issues.
• Symptoms of hemophilia include uncontrolled nosebleeds, bleeding gums, or slow-healing wounds.
  • Symptoms vary in severity.
• Treatments include blood thinners, surgery, or clot-dissolving medications.

Summary of Primary Hemostasis

• Damage to endothelial cells up-regulates von Willebrand's factor from type-4 sub-endothelial collagen.
• Exposed collagen binds circulating von Willebrand's factor (vWF), which binds Glycoprotein 1b receptors (Gp-1b) on platelets.
• 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.
• Fibrinogen is still in precursor form.

Final Stage of Primary Hemostasis

• Thrombin cleaves fibrinogen into fibrin, forming a more stable platelet plug.

Secondary Hemostasis

• Clotting factors that are proteolytically activated activate fibrin, or Factor IA, which forms a fibrin mesh (the cement).
• When Factor X gets activated, it proceeds to activate a common coagulation cascade.

Anti-Platelet Drugs

• Medications prevent blood clots by making blood less sticky.
• Often prescribed to people who have had a heart attack, stroke, or stent placement.
• Examples of anti-platelet drugs are:
  • Aspirin, cyclooxygenase inhibitor
  • Clopidogrel (Plavix), Prasugrel (Effient), Ticagrelor (Brilinta), ADP receptor inhibitors
  • Dipyridamole, adenosine reuptake inhibitor
  • Cilostazol, phosphodiesterase inhibitor