Hemostasis: Vascular Spasm and Platelet Plug

• Hemostasis is the process that prevents blood loss when blood vessels are damaged and stops bleeding in a quick and localized manner

Successful Hemostatic Process

• Speed of reaction is important
• Localization must be at the site of vessel wall injury
• Controllability of the reaction must exist

Hemostatic Processes:

• Vascular spasm
• Platelet plug formation
• Blood coagulation
• Permanent closing of the hole in the vessel

Vascular Spasm

• Occurs instantaneously and lasts for 30 minutes
• Proportional to the vessel wall damage
• Reduces blood loss until other mechanisms can take over
• Mechanisms include nervous reflexes, local myogenic contraction, and vasoconstrictors such as serotonin and thromboxane A₂

Platelet Plug Formation

• Thrombocytes(platelets) are activated by contact with collagen fibers within the subendothelial layers

Thrombocytes

• Round or oval, biconvex disc-shaped cell fragments with a diameter of 2-4 μm
• Thrombocytes lack a nucleus
• Contain granules, mitochondria, rough ER, and ribosomes
• Dense granules contain serotonin, ADP, ATP, and Ca2+
  • Serotonin and thromboxane A₂ are vasoconstrictors
  • ADP causes stickiness
• α granules contain von Willebrand factor (vWF), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), FV, and FXIII
  • vWF acts as a connecting agent
• The average number of thrombocytes is 150 - 400 x 10^9/L
• Thrombocytes have an average lifespan of 8 - 12 days
• Produced in the bone marrow by megakaryocytes through thrombopoiesis, stimulated by thrombopoietin
• Eliminated by the tissue macrophage system in the spleen, liver, and lungs

Substances Released by Platelets

• Platelet-derived growth factor (PDGF) from α-granules is involved in angiogenesis and helps repair damaged vascular walls
• Platelet factor 4 from α-granules is a heparin antagonist
• Von Willebrand factor (vWF) from α-granules helps platelets stick together and adhere to the walls of blood vessels at the site of a wound
• Serotonin from dense granules causes vasoconstriction
• ADP and ATP from dense granules support thrombocyte aggregation
• Ca2+ from dense granules facilitates the activation of many proteins and cytoskeletal rearrangements
• Platelet factor 3 from the cell membrane comprises phospholipids that activate some coagulation factors
• Glycoproteins IIb and IIIa from the cell membrane serve as receptors for fibrinogen and are essential for platelet aggregation
• Thromboxane A2 from the cytoplasm causes vasoconstriction

Steps in Platelet Plug Formation

• Adhesion occurs when platelets stick to exposed collagen, leading to activation
• Change of shape is a result of activation
• Release reaction leads to aggregation and is activated by thromboxane A2, serotonin, and ADP

Function of Platelet Plug

• Important for closing minute ruptures in very small blood vessels, which occur hundreds of times daily
• Occludes the hole in the vessel wall without occluding the vessel itself
• Surface of the activated platelet acts as a cofactor for several enzymatic reactions of the coagulation cascade
• Thrombocytopenic purpura is characterized by hundreds of small hemorrhagic areas under the skin and throughout the internal tissues due to a deficiency of platelets

Endothelial Cells

• Play a significant role in some blood functions
• Create a continuous, non-wetting liner of the inner vessel wall
• Attached to each other by a tight junction with varying degrees of tightness
• Functions include regulation of the coagulation cascade, inhibition of platelet activation, regulation of the permeability of the vessels, involvement in the inflammatory process and its regulation, and control of optimal blood flow through the blood vessel

Substances Produced by Endothelium

• Nitric oxide causes Vasodilation
• Prostacyclin causes Vasodilation, antiaggregation, and antiadhesive effects
• Angiotensin II causes Vasoconstriction
• Endothelin 1 causes Vasoconstriction, proadhesive, and proagregative effects
• Platelet-activating factor (PAF) is involved in the aggregation and degranulation of thrombocytes
• von Willebrand factor (vWf) is a prothrombotic factor and helps platelets stick together and adhere to the walls of blood vessels at the site of a wound
• Tissue activator of plasminogen (tPA) facilitates the conversion of plasminogen to plasmin
• Plasminogen activator inhibitor 1 (PAI-1) inhibits tPA

Blood Coagulation

• Blood drawn from the body thickens into a gel through a cascade of reactions
• Each clotting factor activates the next in a fixed sequence, leading to the formation of a fibrin net
• Blood coagulation only occurs at the site of the injury
• Substances required for clotting include Ca+2, enzymes and factors synthesized by liver cells, and substances released by platelets or damaged tissues

Clotting Factors

• Proteins involved in the process of clotting
• Produced mainly in the liver
• Present in the plasma
• Clotting factors are designated by a Roman numeral and a name
• They can function as enzymes or cofactors

Role of Vitamin K in Clotting

• Vitamin K is required for the synthesis of prothrombin, FVII, FIX, and FX
• Sources include food and bacteria in the intestine
• Vitamin K is a fat-soluble vitamin that is absorbed if lipids are present
• It is inactivated by coumarin, which is used in clinical practice

Coagulation Cascade

• Blood coagulation occurs in vitro in three essential steps
  • Formation of the prothrombin activator
  • Conversion of prothrombin to thrombin
  • Conversion of fibrinogen to fibrin threads
• Fibrin threads trap blood cells and proteins

Extrinsic Pathway

• Damaged tissue cells bear tissue factor (TF; thromboplastin)
• FXa combines with FV to form prothrombin activator in the presence of Ca+2, FVIIa, and phospholipids
• Prothrombin activator forms in seconds (15-20 s)

Intrinsic Pathway

• Activated when blood comes in contact with collagen inside damaged endothelium
• Involves the activation of coagulation factors and platelets
• Platelets aggregate, and phospholipids uncover
• Requires several minutes for production of prothrombin activator

Final Common Pathway

• Prothrombin activator (prothrombinase complex) includes FXa and FVa; the presence of Ca+2 and membrane phospholipids is required
• It catalyzes the conversion of prothrombin to thrombin
• Thrombin converts soluble fibrinogen to insoluble fibrin threads and activates FXIII
• Positive feedback cycle

Fibrin

• Produced from fibrinogen (high molecular weight) by thrombin
• Fibrinogen splits into fibrin monomer and low molecular weight peptides (fibrinopeptides A and B)
• Fibrin monomers polymerize to form fibrin threads
• Fibrin-stabilizing factor (FXIII) creates covalent bonds between the fibrin monomer molecules, resulting in multiple cross-linkages between adjacent fibrin threads
• Fibrin threads adhere to damaged surfaces of blood vessels
• A clot consists of a meshwork of fibrin threads running in all directions and entrapping blood cells
• Fibrin stimulates the formation of granulation tissue and initiates mechanisms leading to the repair of the damaged tissue

Testing Functioning of Pathways

• PT (Quicks test) measures prothrombin time, which is normally 12-15 s and evaluates the function of the extrinsic system
• aPTT measures activated partial thromboplastin time, which is normally 30-40 s and evaluates the function of the intrinsic system
• aPTT and/or PT results can detect a deficiency of involved factors but cannot identify which one
• These tests do not predict bleeding tendency accurately

Limitations of Cascade Model

• FVIII or FIX deficiency leads to bleeding even though the extrinsic and common pathways are normal
• FXI deficiency leads to variable hemostatic deficits with bleeding
• FXII deficiency does not lead to bleeding, despite the requirement of this factor for initiating the intrinsic pathway, but aPTT is prolonged
• The intrinsic or extrinsic pathways alone cannot lead to appropriate thrombus production; an interaction between both pathways is necessary for hemostasis "in vivo"
• This interaction can be explained by the cell-based model

Cell-Based Model

• Involves two surfaces: TF-bearing cells and platelets
• Occurs in three phases
  • Initiation
  • Amplification
  • Propagation

Initiation Phase

• Occurs outside of the vascular compartment on many interstitial cells
• TF and FVIIa form the TF/FVIIa complex
• TF/FVIIa activates FIX and FX
• FXa binds to FVa, resulting in the formation of prothrombinase on the surface of TF-bearing cells
• This leads to the generation of a small amount of thrombin
• Amount of thrombin generated is not sufficient to lead to widespread clot formation

Amplification phase

• Small amount of produced thrombin activates more platelets and their flip-flop mechanisms
• Results in the generation of a platelet procoagulant phospholipid surface
• Also activates endothelial cells
• Cleaves FV, FVIII, and FXI on the platelet membrane to their active forms
• FXIa activates FIX to FIXa
• Platelets become coated in activated coagulation factors, leading to an increased amount of thrombin production

Propagation Phase

• Additional FXa produced due to activity of factors on the thrombocyte surface
• FXa and FVa, along with calcium and a phospholipid (surface of activated platelets), form the prothrombinase complex
• A large amount of thrombin is generated, which is enough to catalyze the reaction of converting fibrinogen to fibrin
• Fibrin structure of the clot is stabilized by covalent bonding under the influence of FXIIIa

In vivo Coagulation

• Intrinsic and extrinsic pathways are not redundant
• Operate on different cellular surfaces to fill different roles
  • Extrinsic - on the TF-bearing (initiating) cells
  • Intrinsic pathway - on platelets

Clot Retraction

• Stabilizes clot
• Actin and myosin in platelets contract within 30-60 minutes
• Platelets pull on fibrin threads, causing clot retraction and expelling serum
• Edges of damaged vessel pulled together

Blood Vessel Repair

• Vessel heals as clot retraction occurs
• Platelet-derived growth factor (PDGF) stimulates the division of smooth muscle cells and fibroblasts to rebuild blood vessel wall
• Vascular endothelial growth factor (VEGF) stimulates endothelial cells to multiply and restore endothelial lining
• Leads to replacement of damaged tissue with a new tissue (other properties than the original)

Fibrinolytic System

• Dissolves small or inappropriate clots and clots at a site of completed repair through fibrinolysis
• Inactive plasminogen incorporated into the clot becomes plasmin, which digests fibrin threads

Clot Prevention

• Smoothness of the endothelium
• Clot formation limited in time and space
• Bloodstream disperses clotting factors
• Antithrombin III with heparin
• Fibrin-antithrombin effect
• Macrophage activity
• Protein C and protein S system (elimination of FVa and FVIIIa)
• Thrombomodulin

Anti-Coagulation Agents

• Suppress or prevent blood clotting
• In vivo: Heparin, released by basophils and coumarin derivatives (antivitamin K)
• In vitro: Stored blood in blood banks treated with citrate phosphate dextrose (CPD) that removes Ca+2
• Thrombolytic agents dissolve clots
• Tissue plasminogen activator (t-PA), Urokinase, and Streptokinase

Inhibition of Coagulation

• Coumarine derivatives act as an antivitamin K, leading to a decrease in production of prothrombin, FVII, FIX, and FX
  • Onset of action is gradual, reaching optimal levels in days
  • Effect is long-term
• Heparin makes a complex with antithrombin III, enhancing its anticoagulant effect
  • Onset of action is very quick
  • Effect is short-term

Thrombosis

• Unwanted intravascular clotting causes Clot (thrombus) formation in an unbroken blood vessel
  • Forms on rough inner lining of blood vessels
  • Can occur if blood flows too slowly (stasis), allowing clotting factors to build up locally and cause coagulation
• Can dissolve spontaneously or dislodge and travel, forming an embolus
• Movable Clot
  • Pulmonary embolus
• Aspirin blocks the synthesis of thromboxane A₂ and reduces inappropriate clot formation