Hemostasis Flashcards
What is hemostasis? What are the two outcomes of an out of balance hemostatic system?
prevention of blood loss upon injury to the vessel and reestablishment of blood flow; hemorrhage and thrombosis
What 5 things cause a thrombosis?
blood flow or hemodynamics, blood vessel wall, blood constituents, genetic, and aquired
What 4 things can cause a hemorrhage?
synthesis or function of clotting and anti clotting factors, generation and function of platelets, genetic diseases, acquired
What are the three functional attributes of the hemostatic system?
self-aware and everywhere, self sustaining, autoregulation
What is a thrombus?
abnormal clot that develops in a blood vessel
What is an embolus?
a clot that flows freely in the blood
What is an infarct?
necrotic tissue area formed due to obstruction of blood due to an embolus or a thrombus
What are the components of the hemostatic system?
platelets, plasma, blood vessel constituents: endothelial cells, basement membrane, smooth muscle cells and fibroblasts, Tissue/cells where clotting are synthesized and degraded
what triggers and what are the steps of the hemostatic cascade?
injury or pathology disturbs endothelial cells, basement membrane components are exposed; vasoconstriction, platelet adhesion and aggregation, coagulation, blood vessel repair, and clot dissolution
Describe the set up of the vessel wall inside to out.
smooth surface endothelial cells, basement membrane, ECM containing fibroblasts, and smooth muscle cells surrounding
What is the first stage of platelet activation and plug formation?
initiation and activation, adhesion of platelets to thrombogenic substrates or injured vessel wall components, this activates platelets and induces shape changes and signaling cascades
What is the second stage of platelet activation and plug formation?
extension: release of calcium, ADP, TXA2, thrombin, this recruits additional platelets to the site of injury
What is the third stage of platelet activation and plug formation?
perpetuation: events that stabilize platelet to platelet adhesion resulting in formation of the platelet plug
Name the adhesion receptors on platelets and what they interact with.
GP-VI binds with collagen, GP1b-IX-V binds with vWF, GPIb/IIa (alpha 2/ beta 1) mediates binding with collagen, GP IIb/IIIa (alpha IIb/ beta III) mediates adhesion between platelets via fibrinogen, GPCR Gs binds with PGI2, GPCR Gq binds with TXA2, uPAR Gq binds with Thrombin, P2Y1 Gi and P2Y2 Gq binds with ADP, GPCR PAR 1 and PAR 2 both bind to thrombin
What happens when GP-VI binds to exposed collagen?
initiates a signaling cascade to activate platelets
What happens when vWF binds to GP1b-IX-V?
after undergoing a change vWF has an increased affinity for GP1b-IX-V, upon forming the GP1b-IX-V-vWF complex binds to collagen which initiates platelet adhesion to the exposed vessel surface
What else does vWF do besides bind GP1b-IX-V?
some remains bound to the basement membrane, vWF transports and sequesters coagulation factor VIII
What does integrin alpha 2 beta 1?
mediates adhesion of platelets to collagen on the exposed vessel surface
What does integrin alpha IIb beta 3?
mediates adhesion between platelets via fibrinogen
Where is vWF made and stored?
synthesized by endothelial cells and megakaryocytes and are stored in the platelets and endothelial cells
Which receptors and their substrates activate platelets in the coagulation cascade?
ADP activates via receptors P2Y1(Gi) and P2Y2(Gq), thrombin a protease binding with PAR1 and PAR2, and TXA2 which is coupled with Gq or Gi
Which receptors and their substrates inhibit platelet coagulation?
PGI2 and is coupled to Gs
Describe the steps of initiation of the clotting cascade.
hemodynamic changes induce a conformational change in GP1b-IX-V increasing its affinity for vWF, their binding triggers a signaling cascade, this permits the initial adhesion of rolling platelets to collagen via vWF, adhesion of platelets to collagen is strengthened and stabilized thru alpha2beta1 and GP-VI, this activates platelets and initiates a shape change and forces them to exocytose stored TXA2, ADP and thrombin
Describe the extension and perpetuation of the platelet plug formation.
TXA, ADP and thrombin are released by activated platelets on collagen bed, they bind to GPCRs on circulating platelets nearby activating them and recruiting them to the site of the injury where the new platelets adhere to the other platelets by crosslinking of alphaIIbeta3 via fibrinogen forming the hemostatic plug
Negatively charged phospholipids translocate to the outer membrane leaflet of platelets why?
allows the assembly of the tenase complex and the prothrombinase complex which assures platelet aggregation and activation as well as clotting cascade initiated and restricted to site of injury or are constrained locally
What do pro-coagulation factors do?
activated temporarily upon injury or in disease states, end point is to produce thrombin which coagulates blood via changing fibrin into fibrinogen
What do anti-coagulation factors do?
prevent clotting and/or dissolve clots; most dedicated to inhibiting thrombin activity or destroy factors that facilitate the production of thrombin
What are the four categories or classes of clotting and anticlotting factors?
enzymes, cofactors, control proteins, and substrates
What are some common properties and examples of enzyme clotting and anticlotting factors?
circulate in inactive form (zymogens or proenzymes) bound to serpins, serine proteases, activated via proteolytic cleavage, contain 3 domains GLA 9undergoes gamma carboxylation, Kringle (regulatory), and the Catalytic domain; Prothrombin, Protein C, Prekallikrein, Factor VII, IX, X, XI, XII, & XIII
What are some common properties and examples of cofactor clotting and anticlotting factors?
bind and stabilize clotting factors but have no enzyme activity; Tissue factor, Thrombomodulin, Protein S, HMWK, Factor V and VIII
What are some common properties and examples of control protein clotting and anticlotting factors?
regulate coagulation and anticoagulation; antithrombin, Protein C, alpha1 anti trypsin, Heparin II, TFPI, and alpha2 macroglobulin
What are some common properties and examples of substrate clotting and anticlotting factors?
fibrinogen and fibrin
How is thrombin produced?
Prothrombin undergoes cleavage via prothrombinase
Where are most clotting factors created and what type of molecule are they?
the liver but a few by monocytes, endothelial cells and megakaryocytes, they are mostly glycoproteins
List the extrinsic pathway of coagulation.
Tissue factor is exposed on fibrblasts or released locally, factor VII is activated by thrombin and binds with TF, Phosholipds(-) on platelet surface with Ca, this complex with XIa (activated by thrombin) activates IX to IXa which binds with VIIIa (activated by thrombin) PL and CA to form the tenase complex, which activates V to Va and in combination with TF-VIIa-PL-Ca actiates X to Xa, Xa and Va bind to PL and CA on platelet surface forming the Prothrombinase complex
List the intrinsic pathway.
the blood contacts a negative charge and form contact factor complex composed of XIIa, high molecular weight kininogen (HMWK) and prekallikrein (Pre-K); this activates XI to XIa which activates IX to IXa which binds with VIIIa (activated by thrombin) and PL and CA to form the Tenase complex which activates X to XA which binds with Va (activated by thrombin) PL and Ca on platelet surface forming the prothrombinase complex
What are the three steps in the change of fibrinogen during clotting?
enzymatic conversion to insoluble fibrin monomer by thrombin, aggregation of fibrin monomers to form the soft clot, and cross-linking of aggregated fibrin monomer by XIIIa a transglutaminase forming the hard clot
What changes occur in fibrinogen to get fibrin?
4 peptides (2FP and 2 FPB) on the N-termini carry a - charge and keep fibrinogen soluble are cleaved
What is clot retraction?
the fibrin meshwork intertwining the platelets traps monocytes and RBCs, begins to condense into a compact structure, first part of repair process
What occurs after clot retraction?
platelets attached to fibrin meshwork contract via contractile proteins (actin and myosin) and pull clot (retraction), the clot condenses by squeezing out a fluid called serum
What is fibrinolysis? Describe the process.
lysis of firbin clot, clot is dissolved by plasmin (ser protease) which digests corss-linked fibrin monomers
How is plasmin activated?
from plasminogen via Plasminogen activator either t-PA (endothelial cells secrete) or urokinase PA (u-PA, synthesized and secreted by urinary tract epithelial cells, monocytes, and macrophages), can also be activated by streptokinase secreted by beta-hemolytic streptococci; produces D dimers and E-fragments
What are D dimer used for clinically?
a clinical test to rule out deep vein thrombosis and pulmonary embolism
what are the four classes of anti-coagulating factors?
Class A: tissue factor pathway inhibitor (TFPI), Class B: Ser Protease inhibitors (Serpins) inhibit thrombin and other factors through binding antithrombin III (ATIII) & Heparin cofactor II
Class C: inhibit formation of prothrombinase Protein C & S
Class D: inhibitors of tPA and Plasmmin, plasminogen activator inhibitor 1 (PAI-1) and plasmin inhibitor (Antiplasmin)
How does TFPI work?
produced by subendothelium and circulates as a complex of lipoproteins, Complex of factor VIIa and TF ctivqtes IX and X, when the concentration of Xa increases beyond threshold, Xa binds to circulating TFPI and activates it, TFPI inhibits TF-VIIa complex, prevents generation prothrombinase, Autoregulatory aspect
Where is ATIII made, type of molecule, and how does it work?
glycoprotein synthesized in the liver, half life of 3 days, inactivates thrombin by covalently binding to it
What is the role of Heparin or Heparin sulfate?
binds at endothelial surface with thrombin and ATIII augmenting ATIIIs inhibition by 1000x, released by mast and endothelial cells
How does heparin cofactor II work?
similar mechanism as ATIII, covalently binds thrombin then binds to heparin
How does Protein C and Protein S work?
vitamin K dependent, C has enzymatic activity, circulates as zymogen; S is cofactor produced in liver; thrombin produced in sufficient quantity activates Protein C in complex with Thrombomodulin (expressed on endothelial surface) and Protein S, once activated C destroys co-factors Va and VIIIa
How does PAI-1 work?
t-PA and u-PA circulate in complex with PAI-1, once they get trapped in a clot they dissociate, this allows plasmin to only by generated in the clot, after the clot is dissolved free t-PA is cleared in the liver (half life 5-6min) (auto regulation)
How does fibrin affect t-PA?
in the absence of fibrin t-PA has a low affinity for plasminogen, when bound to fibrin the t-PA affinity is 50-100x more decreasing Km, allows fibrinolytic reactions to only occur on the surface of the clot (auto regulation)
what does alpha2-antiplasmin do?
a serine protease inhibitor, binds to free plasmin preventing it from digesting fibrinogen and clotting factors, fibrin in a clot reduces the affinity of antiplasmin for plasmin (auto regulation)
Where are most coagulating factors synthesized? Examples.
Prothrombin, fibrinogen, and factors VII, IX, X, XI, protein S and C
Vitamin K is a cofactor for an enzyme that does what? What coagulation factors does it act on?
gamma carboxylation of acid residues of coagulation factors (GLA), Prothrombin, VII, IX, X, XI, XII and protein C and S
Where does Ca bridge in the Tenase and Prothrombinase complexes?
between the PL on activated platelets and gamma-carboxylated GLA on clotting factors
If vitamin K is absent what happens?
no gamma-carboxylation so clotting factors are unable to bind to platelets and therefore remain inactive
what is Virchows Triad for hemostasis?
hemodynamic alterations (turbulence or stasis), endothelial dysfunction (valvular disorder, atherosclerosis), and hypercoagulability (drugs too)
What are the two main categories of hemostatic disorders and their subcategories?
Platelets (activation, adhesion, & aggregation) and coagulation factors (pro-clotting, anticlotting)
What are the clinical manifestations of hemostatic disorders?
Cutaneous, mucosal blisters, GI bleeding, cerebral hemorrhage, intramuscular bleeding, muscle hematomas
What is hemophilia?
x linked hemostatic disorder, a is factor VIII deficiency, B is Factor IX deficiency coagulation time prolonged, C is XI deficiency coagulation time prolonged
what is factor XIII deficiency?
rare genetic deficiency, umbilical stump bleeding, spontaneous intracranial hemorrhage, die young; only form soft clots
What are the three types of fibrin disorders?
afibrinogenemia (none), hypofibrinogenemia (low) results in only soft clots, dysfirbinogenemia (dysfunctional) firbin level low, dysfunctional due to mutatations, all chains effected, soft clots only
what is von Willebrand disease (VWD)?
autosomal dominant, 1% pop, loss of function: adhesion of platelets to vessel impaired due to many factors, prolonged bleeding time, spontaneous hemorrhage most common; gain of function: platelet adherre to wall without injury, in VWD IIb vWF has increased affinity for GPIb-IX-V and adheres to wall spontaneously, weak adhesion, platelets washed away easily
what is factor V deficiency look like?
leads to mild hemophilia, mutations confer resistance to destruction or increase half life, associated with thrombophilia ex: vein thrombosis
What does protein C and protein S deficiency look like?
in deep vein thrombosis and Purpura fulminans; life threatening severe clotting throughout body, ultimately causing death of tissues, due to homozygous deletion of protein C; S deficiency predisposes individuals for thrombosis
Deficiency in what clotting factor is seen in 2% of patients with venous thromboembolic disease?
ATIII, inherited autosomal dominant, deficiency results from mutations that affect synthesis or stability of ATIII or from mutations that affect the protease or heparin binding sites of ATIII
Where do antiadhesion antiplatelet agents work?
Von Willebrand Factor Inhibitors; vWF binding to GPIb-IX-V
Where do anti-activation antiplatelet agents work? examples.
inhibitors of signaling; TXA2-Aspirin, ADP receptor-dipyridamole, Collagen receptor- Clopidogrel, Thrombin inhibitors- Ticlopidine
where do anti-aggregation antiplatelet agents work? Examples.
anti-GPIIbIIIa (alpha 2b beta 3); Monoclonal antibodies- (ReoPro, c7E3, abcixima), synthetic peptides- (MK-852, MK-383, Ro44-9883, eptifibatide), integrin alpha2bbeta3 inhibitor, nonpeptide receptor agonist- tirofiban
what are the classes of GPCR targeted drugs, method of action and examples?
activation inhibitors- ADP receptor antagonists (Ticlopidine, Clopidogrel, Prasugrel, Tigogrelor) or PAR-1 antagonists (SCH530348 and E5555), Selective generators-PGI2, and Thromboxane inhibitors- TXA2 (Aspirin, Ridogrel, Picotamide, Ramatroban)
How do NSAIDs affect the hemostatic system? Examples.
affect prostaglandin synthesis by blocking prostaglandin synthase or cyclo-oxygenase (COX1 and COX2) which are needed to synthesize TXA2; Aspirin, indomethacin, ibuprofen)
How does Aspirin inhibit COX 1 and COX 2?
covalent acytelation of serin residues of the active site
How is TXA2 recovered following NSAID use?
endothelium makes more enzyme, platelets have no nucleus and require production of new platelets (5-7 days)
what are the different categories, method of action and drugs listed in each for anticlotting factor drugs?
Direct: Direct Thrombin inhibitors which bind to thrombin and inhibit activity (Hirudin, Argatroban, Melagatran) and Indirect Thrombin Inhibitors which inhibit thrombin generation (AntiXa antibodies, TFPI, Soluble Thrombomodulin) or Indrect: ATIII dependent which increase ATIII activity (Heparin, Heparin Fragments) and Vitamin K Anatagonists which inhibit synthesis of clotting factors (Warfarin, Coumarin, Dicoumarol)
How does Warfarin work? Other names?
inhibit vitamin K dependent carboxylase responsible for gamma-carboxylation reactions needed for thrombin, VIIa, IXa, Xa, XIa and XIIa, also inhibits vitamin K dependent Protein C and S; inhibit pro-clotting and anticlotting (Coumadin, Jantoven, Marevan, Uniwarfin, Warf)
How do omega 3 fatty acids affect the hemostatic system?
EPA (omega 3) produce PGI3 instead of PGI2 and TXA3 instead of TXA2; PGI3 has same antiplatelet activity as PGI2 but TXA3 has much weaker activity than TXA2
