Biochemistry of clotting Flashcards
1. Describe the basic biochemical and physiological events that lead to thrombosis 2. Describe the endogenous thrombolytic system. 3. Describe how antithrombin and protein C&S act as natural circulating anticoagulants in the context of the coagulation cascade 4. List the laboratory tests used to monitor coagulation and understand their biochemical basis
How to obtain plasma
- collect blood into an anticoaglant
- red blood cells, white blood cells and platelets removed (centrifuge giving pellet = cells and supernatant = plasma)
Clotting factors in plasma
Still in their inactive (zymogen) forms
How to obtain serum
- Allow blood to clot
- clot separated into a fibrin clot (fibrin plus trapped cells) and serum - centrifuge into pellet (fibrin + trapped cells) and supernatant (serum)
Clotting factors in serum
Have been activated and fibronogen has been depleted
Hoemostasis blood (goal + players)
Mechanisms that ensure retention of blood within the vascular system, involve:
- endothelial cells lining the blood vessels
- platelets
- clotting factors
Virchow’s triad of normal homeostasis
1) Normal vascular epithelium normal blood flow
2) Normal blood flow correct homeostatic balance (pro-coagulant, anti-coagulant, fibrinolysis)
3) Normal vascular endothelium correct hemostatic balance
Platelets -formation
Formed from megakaryocytes (WBC synthesized in the marrow)
Characteristics of platelet
Non-nucleated
2-3 um diameter
Half life platelets
5-9 days
Activation of platelets
-by thrombin or ADP
Physiological events leading to clot formation (thrombosis)
1) Damage to endothelial cell layer
2) Platelet plug formation (platelets adhere to site of endothelial cell injury -platelets aggregates and mass enlarges)
3) Fibrin clot formation - fibrin deposition stabilizes the platelet thrombus
* *activated platelets = the surface where blood will clot
thrombolysis
Tissue repair and fibrin clot dissolution
Challenges of hemostasis
1) blood must remain fluid most of the time
2) blood clotting must occur rapidly
3) Clot must be formed at site of injury in the flowing blood
4) Clot must be readily dissolved
MOA of fibrinolysis/thrombolysis
Fibrin polymer (insoluble blood clot) + plasmin (a protease) = fibrin degradation products (also called D2E fragments or D-dimers --> means have clotted and are now in the process of dissolving the clot --> a marker of thrombosis)
What is the extrinsic pathway, what is the intrinsic pathway
Extrinsic pathway -involves tissue factor (only found in blood when there has been tissue damage i.e. normally extrinsic to blood)
Intrinsic pathway = all proteins involved found in blood
Both pathways need a platelet surface (reactions in the middle)
Share a common pathway (thrombin becoming cross-linked fibrin)
After surge of thrombin activity want clotting cascade to take over –> Tissue Factor Pathway inhibitor inhibits enzymes in extrinsic pathway
-thrombin able to go back and activate f
Propagation
-intrinsic pathway
Fibrinogen
- made up of 6 polypeptides (alpha2beta2gamma2)
- three globular domains linked by triple helixes (shaped like a dumbell)
MOA Fibrin formation
- via proteolysis
- thrombin cleaves 4 peptide bonds in fibrinogen
- gives 4 fibrin monomers (soluble)
MOA fibrinogen activation
New end terminus in central domain interacts with terminal end of another monomer (so get staggered polymerization)
Polymerization = spontaneous polymerization of fibrin monomers (to make unsoluble fibrin polymer)
-cross linking (introduced by factor XIIIa –> covalent bond between adjacent terminal domains- two covalent cross link/fibrin unit)
-gives tough insoluble fibrin clot
Importance of vitamin K to clotting
-some clotting factors require vitamin K-dependent carboxylation during their biosynthesis to be biologically active
Origin of vitamin K
- found in vegetable oil (fat-soluble) and green leafy vegetables
- synthesized by gut flora
- normaly gut bacterial synthesis is sufficient
Vitamin K-dependent carboxylation
Some glutamic acid residues are carboxylated to gama-carboxyglutamic acid (gla)
i.e. glu-peptie –> gla -peptide
Action of Gla proteins
1) Gla binds calcium ions leading to a conformation change
2) Ca2+-Gla proteins can bind to phospholipid membranes (supplied by activated platelets at the site of injury)
3) Ensures that fibrin formation ccurs at the site of injury instead of in the flowing blood
Non-carboxylated vitamin K-dependent proteins
Biologically inactive
Gla-proteins and platelet plug
-only Ca2+-Gla proteins bind:
factor VII, X, IX, prothrombin (the vitamin K proteins)
-factors Va and VIIIa also bind (not via Gla though)
Complex =
factor IXa, Factor 10, prothrombin (zymogen)
Function of compexes bound to platelet membrane surface
Allow for
a) concentration
b) orientation
- factor 9 a binds in a way that can cleave factor 10
- factor 10a binds in way can cleave PT
c) localization
- at site of injury where phosphatidyl serine has been exposed
Can make much more thrombin in this complex!! (empahsizes importance binding proteins, phospholipid membrane surface and Ca2+ ions)
Symptoms of Factor XII deficient patients
Do not bleed
Thrombomodulin Protein-C- Protein S Pathway
Thrombomodulin appears on endothelial cell plasma membrane (at site of injury)
Converts thrombin from Procoagulan to an anti-coagulant
(converts activity of thrombin)
Protein C is anticoagulant and in presence of protein S inactivates Factor Va, VIIIa
Plasminogen activation
Plaminogen converted to plasmin via plaminogen activator (a protease)
Role of plasmin
Able to cleave a fibrin clot in triple helix region
Gives D-dimers (D2E fragment or fibrin degradation product) - these are soluble and cleared by the liver (diagnosit cmarekr for thrombosis)
Laboratory Coagulation Tests
1) Prothrombin Time (PT)
2) Activated partial thromboplastin time (APTT) - also called Partial thromboplastin time
Thromboplastin
A crude brain extract of phospholipid & tissue factor
Partial thromboplastin
Consists of phospholipid but no tissue factor
Prothrombin time
-activate with thromboplastin
-add Ca2+
-measure clotting time
Measures extrinsic and common pathway
International normalized ratio
-prothrombin time for a normal individual varies with country
INR = (PT/PTref)x ISI
Activated Partial Thromboplastin time
- incubate blood with kaolin
- add partial thromboplastin (phopholipid but no tissue factor)
- add Ca2+
- measure clotting time
- measures intrinsic and common pathway
