APPP 17 and 22: Blood-Platelets and Hemostasis Flashcards
What is hemostasis?
series of processes aimed at preventing excessive blood loss after an injury
- cessation of blood loss from a damaged vessel
What does successful hemostasis depend on? (3)
- vessel wall
- circulating platelets
- plasma-coagulation proteins
Briefly describe the process of hemostasis.
- starts with constriction of blood vessels to limit blood loss
- followed by contribution from platelets and coagulation proteins to form a thrombus (blood clot)
What is a thrombus?
clot that adheres to the interior wall of an artery or vein
What is an embolus?
blood clot that has been dislodged and is travelling throughout the bloodstream
Why are both thrombi and emboli dangerous?
may occlude blood vessels, obstruct flow of blood through circulatory system, and deprive tissues of oxygen and nutrients
What is thrombosis?
formation of an unwanted blood clot in the artery or vein
What is arterial thrombosis?
usually a result of adherence of platelets to the arterial wall
- largely a phenomena of platelet activation
- arterial thrombi are pale, granular, and have a lower cell count
What can arterial thrombosis lead to? (5)
- ischemia
- myocardial infarction
- angina
- stroke
- peripheral arterial disease
What is the management strategy for arterial thrombosis?
mainly focused on anti-platelet strategies
What is venous thrombosis?
largely a consequence of activation of the clotting (coagulation) system and hence are made up of RBC and fibrin
- venous thrombi are soft, gelatinous, deep red, and have a higher cell content
- develops in areas of stagnated blood flow (especially in deep leg veins, most commonly femoral vein, near venous valves)
What are the causes of deep vein thrombosis? (4)
- surgery
- injury – ie. broken leg
- sitting or inactivity
- birth control
What would happen if the venous thromboemboli dislodges from the deep veins of the leg?
travels to right side of the heart to reach the lung, where it causes pulmonary embolism (potentially life-threatening)
What is the management strategy for venous thrombosis?
mainly centred on anticoagulation strategies
Arterial Thrombosis
What does clot formation require?
- platelet adhesion, activation, and aggregation
- formation of thrombin
Arterial Thrombosis
What is thrombin and its function?
enzyme that catalyzes the production of fibrin which (when cross-linked) stabilizes the clot
Arterial Thrombosis
What happens in response to an injury to a blood vessel?
- local vasoconstriction occurs due to local spasm of the smooth muscle in the wall of the blood vessel as a result of secretion of vasoconstrictors such as endothelin-1
- immediately after vasoconstriction, primary hemostasis occurs – requires platelets (provides initial plug at sites of vascular injury)
What are platelets?
anucleate, cellular fragments that circulate as inactive, non-binding concave discs
Platelets perform a very important biologic role in the vascular system. Describe this.
- interaction between platelet and vessel wall is important
- platelets normally do not adhere to healthy arterial walls, and are not activated by the vascular endothelium – because platelets have coated glycoproteins which carry negative charges and are therefore repelled by the endothelial cell (EC) which also has a glycoprotein coat of negative charge
- damage to blood vessels exposes collagen and other underlying tissue (sub-endothelium) which have positive charges
- therefore at the site of injury (damaged vessel wall), platelets begin to adhere to the damaged site within seconds, then platelet activation (release of intracellular granules) and platelet aggregation occurs to form a platelet plug
Platelet Adhesion
When does it occur and why?
within seconds of vascular injury, platelets firmly adhere to collagen fibrils in the vascular sub-endothelium via ionic interaction and specific receptors
- this process is provoked by the loss of an intact endothelial lining of the blood vessel, as well as by the exposure of the platelet to activating sub-endothelial structure (ie. collagen)
Platelet Adhesion
How do platelets adhere to collagen in vascular sub-endothelium?
via a specific platelet collagen receptor made up of glycoproteins (Gp)
Platelet Adhesion
What is GPVI?
central receptor that facilitates direct contact with sub-endothelial collagen
Platelet Adhesion
What are the other receptors also suggested to play a role in contacting platelets directly to collagen?
α2β1-integrin and GPIa
Platelet Adhesion
What does GPIb do?
can indirectly interact with collagen via its binding to the von Willebrand factor (vWF)
Platelet Adhesion
What is von Willebrand factor (vWF)?
unique adhesive glycoprotein that is released from both the injured endothelial cell and platelets, which allows platelets to remain attached/anchored to the vessel wall despite the high shear forces generated within the vascular lumen
Platelet Adhesion
What does vWF bind to?
both GPIb on the platelet membrane and to the exposed collagen (‘bridging action’)
- in this way, platelets have an additional mechanism by which they get anchored to the site of the injured endothelium
Platelet Activation
Briefly describe this process.
initial adherent platelets undergo a process of activation (or degranulation) – on binding of platelets to underlying collagen (most important stimulus for platelet activation) or vWF, there is Ca2+ mobilization from intracellular stores (within DTS-dense tubular system) into the cytoplasm, which increases the amount of Ca2+
Platelet Activation
What is the most important stimulus for platelet activation?
binding of platelets to underlying collagen
Platelet Activation
What can Ca2+ promote?
platelet shape change
- platelets contain contractile proteins like actin and myosin
- allows for movement of granules
Platelet Activation
What happens after Ca2+ increases and granule movement begins?
pre-packaged platelet granules that contain second wav agonists (like TXA2 and ADP) are released (degranulation)
- released ADP and TXA2 can further enhance platelet activation by binding to their respective receptors (P2Y12 receptor and TP receptor) to increase intracellular Ca2+ even more in these ‘frontline’ platelets, or activate ADP and TXA2 receptors on other secondary platelets (and therefore augment their intracellular Ca2+)
- this robust increase in Ca2+ stimulates cyclooxygenase to promote the synthesis of thromboxane A2
Platelet Activation
How is thromboxane A2 synthesized?
- Ca2+ activates phospholipase A2, which cleaves platelet membrane phospholipids and liberates arachidonic acid
- in the presence of cyclooxygenase, arachidonic acid forms prostaglandin H2 (PGH2)
- metabolism of PGH2 is facilitated by thromboxane synthase to produce thromboxane A2 (TXA2)
Platelet Activation
What are second wave agonists?
TXA2 and adp
- generate an additional increase in intracellular Ca2+, likely through release from intracellular stores
Platelet Aggregation
What promotes platelet aggregation?
second wave chemical mediators promote platelet aggregation by exposing platelet surface receptors (that are normally inactive on resting platelets but undergo conformational transformation when there is an increase in Ca2+)
Platelet Aggregation
What is the predominant receptor? What does it do?
GPIIb/IIIa
Platelet Aggregation
What does GPIIb/IIIa do?
- facilitates contact with circulating proteins, mainly fibrinogen (but also von Willebrand factor)
- fibrinogen can then act as a bridge between two platelets, which are essential in the process of adhesion and aggregation (RGD (arginine-glycine-aspartic acid) amino acid recognition sequence on fibrinogen makes this possible – each fibrinogen has 2 RGD sequences)
Platelet Aggregation
What is critical for platelet aggregation?
fibrinogen:GPIIb/IIIa interaction
- platelet-fibrinogen linkages rapidly enlarge the platelet plug (called the primary hemostatic plug)
- primary hemostasis is the name given to this process of platelet-plug formation at sites of injury – occurs within seconds of injury and is of prime importance in stopping blood loss
Is the platelet plug stable?
NO – can be dislodged
- therefore the process of secondary hemostasis begins, and requires several minutes for completion
Describe the process of secondary hemostasis.
- begins with local activation of plasma coagulation factors (ie. thrombin)
- eventual formation of a fibrin clot that strengthens the primary hemostatic plug
In the coagulation cascade, what does each stage involve?
the conversion of a precursor protein (synthesized in the liver that is normally inactive) to an active protease (indicated by ‘a’) by cleave of one or more peptide binds on the precursor molecule
What are the usual components involved in each stage of the coagulation cascade?
- protease from the preceding stage
- precursor protein
- non-enzymatic protein co-factor (reaction accelerator)
- Ca2+
- organizing surface (ie. phospholipid surface of activated platelets in vivo)
What are the coagulation proteases of the coagulation cascade?
- HMWK (high molecular weight kallikrein)
- prekallikrein
- factors XII, XI, IX, X, VII, and II (prothrombin)
What are the non-enzymatic protein co-factors of the coagulation cascade?
- factors V, VIII
- tissue factor (TF or thromboplastin) – glycoprotein receptor found on the surface of a number of cells surrounding blood vessels, present on extravascular tissue
What are the final events in the coagulation process?
- conversion of prothrombin to thrombin by factor Xa
- conversion of fibrinogen (soluble) to fibrin (insoluble) monomer in the presence of thrombin (aka factor IIa)
What are the 2 major pathways of the coagulation phase?
- intrinsic system – slower (several minutes)
- extrinsic system – faster (within seconds)
How is the intrinsic system initiated?
- all of the clotting factors are present within the blood vessels
- factor XII is activated on contact with a negatively charged surface (ie. glass) or prolonged exposure to negative charges on endothelial cells
- this initiates the intrinsic coagulation system
How is the extrinsic system initiated?
- initial stimulus (tissue factor or TF) is present outside the blood vessels
- damage to blood vessels exposes TF-containing cells from underlying layers to the bloodstream
- TF is then able to bind in the presence of Ca2+ to factor VII, which circulates in the bloodstream – TF acts as a high affinity receptor for factor VII)
- this sets off sequential protease activations
Where is tissue factor (TF) expressed?
- proposed to be a cell surface glycoprotein that is membrane-bound
- under pathological conditions, also expressed on circulating leukocytes and possibly activated endothelial cells
- present on microvesicles (MVs), which are small membrane vesicles released from activated cells – fragments of plasma membrane from various cell types including platelets and leukocytes)’- these intravascular sources of TF may trigger formation of venous clots
What happens when the intrinsic and extrinsic pathways converge at a final common pathway?
leads to generation of thrombin, fibrinogen, and formation of insoluble fibrin
- fibrin undergoes covalent cross-bridging between adjacent fibrin monomers
- fibrin ‘mesh’ of cross-linked fibrin monomers can be seen as white string-like substance trapping RBCs in a fresh clot
- RBCs do not stick together – they are held together by fibrin
What happens when thrombin binds to thrombomodulin expressed on EC surfaces?
prevents it from cleaving fibrinogen
- instead, it cleaves and activates protein C (in the presence of protein co-factor Va) which inhibits clotting by cleaving and inactivating two pro-coagulant factors Va and VIIIa
What is protein C and what does it do?
natural plasma protein
- together with its co-factor protein S (which results in activated protein C), it degrades factors Va and VIIIa and therefore provides natural anticoagulation by inhibiting activation of factor X and prothrombin
What is antithrombin?
an endogenous anticoagulant
Venous Thrombosis
In deep vein thrombus formation, what 3 factors contribute to formation of a thrombus?
- reduced blood flow and stasis (associated with surgery, hospitalization, paralysis, long-haul travel, pregnancy, etc.)
- thrombophilia or procoagulant changes in blood (where body makes too much off a blood clotting protein like prothrombin or a blood clotting protein like factor V does not function well – cannot be inactivated by protein C and S)
- activation of the endothelium
Venous Thrombosis
What does the presence of valves in veins lead to?
turbulent flow and reduced oxygenation of the valve endothelium that may activate the endothelium and allow a thrombus to form
- this is because the valve pocket sinus can lead to surface expression of adhesion proteins (P-selectin, E-selectin, vWF) due to its tendency to become hypoxic
Venous Thrombosis
What do most circulating leukocytes express?
P-selectin glycoprotein ligand-1
Venous Thrombosis
What does PSGL-1 do?
also secretes microvesicles (MV) that express PSGL-1 receptors and tissue factor (TF)
Venous Thrombosis
Describe the process.
- leukocytes and MVs bind to the activated endothelium via PSGL-1
- bound leukocytes become activated and express TF
- by binding to factor VII (and activating it to VIIa) to form TF-FVIIa complex, there is activation of factor X to Xa
- this initiates the coagulation process where large amounts of thrombin are generated
- local activation of the coagulation cascade overwhelms the protective anticoagulant pathways and triggers thrombosis
Venous Thrombosis
What happens once a thrombus is formed?
the thrombus may extend along the vessel and embolize
Venous Thrombosis
What do valve pocket thrombi contain?
mostly red cells and fibrin
