Hematology (Week 2--Howard) Flashcards
Normal lab values
Total bilirubin: 0..1 - 1.0 mg/dL
Direct (conjugated) bilirubin: 0 - 0.3 mg/dL
Ammonia: 15 - 45
AST: 8 - 20 U/L
ALT: 8 - 20 U/L
PT (prothrombin time): 11 - 15 sec
Platelet count: 150,000 - 400,000 /mm3
Composition of hemoglobin
4 molecules of globin (2 alpha; 2 beta)
4 molecules of heme (each has protoporphyrin IX and Fe2+)
From RBC to bilirubin
RBC engulfed by macrophage –> heme broken down to biliverdin –> biliverdin broken down to bilirubin –> bilirubin (insoluble) binds albumin in plasma and becomes soluble –> complex travels through blood to liver –> liver conjugates bilirubin with glucuronic acid to solubilize bilirubin –> (conjugated bilirubin goes from liver to gallbladder) –> conjugated bilirubin secreted in bile into intestine –> in intestine, glucuronic acid removed and bilirubin converted to urobilinogen –> most urobilinogen remains in intestine, is oxidized by intestinal bacteria to brown stercobilin; some urobilinogen reabsorbed and enters portal blood and some of that urobilinogen transported from blood to kidney where is converted to yellow urobilin and excreted
When does jaundice occur?
Jaundice occurs when serum bilirubin exceeds 3mg/dL
Causes of increased unconjugated bilirubin
Hemolysis
Liver disease
Causes of increased conjugated bilirubin
Liver disease resulting in hepatocyte swelling and/or canaliculi damage
Extrahepatic obstruction of biliary duct (gallstones)
Total, direct, indirect bilirubin
Direct: conjugated bilirubin
Indirect: unconjugated bilirubin
Indirect = total - direct
Common causes of jaundice in neonates
Physiological jaundice: develops 2nd or 3rd day of life; because liver cannot yet conjugate bilirubin (get high unconjugated bilirubin)
Blood type (ABO or Rh) incompatibility: causes RBC hemolysis and (high unconjugated bilirubin); mother Rh negative has Rh antibodies that attack/lyse baby’s Rh positive RBCs
Less common: Glucose-6-PO4-dehydrogenase deficiency (hereditary hemolytic disorder); Gilbert’s diesase (hereditary reduction of bilirubin conjugation)
Kernicterus
Brain damage in newborn due to deposition of unconjugated (insoluble) bilirubin in brain (associates w/membrane lipids and interferes with membrane function)
When >20mg/dL unconjugated bilirubin in blood
Prevented by phototherapy (blue lame in birthing room), which causes bilirubin to be photoisomerized to water-soluble products excreted in bile or urine
Synthesis of heme
Succinyl CoA + glycine converted to ALA by ALA synthase –> ALA converted to porphobilinogen by ALA dehydratase –> 4 porphobilinogens combine to form protoporphyrinogen IX –> –> heme
What happens if VERY first step in the liver (ALA synthesis) is defective?
Lethal!
Where is heme biosynthesized?
Most cells (heme is present in cytochromes and other substances)
Regulation of heme biosynthesis best understood in liver
Primary porphyria
Genetic defect in heme synthesis pathway that causes buildup of heme precursors
Acute porphyria (neuroviscreal or hepatic porphyria): defect in early step; abdominal pain, constipation, vomiting, paralysis, neuropsychiatric disorders; accumulation of toxic ALA and porphobilinogen and deficiency of heme in neurons
Cutaneous porphyria (erythropoietic porphyria): defect in late step; sun light-induced skin lesions, urine and teeth turn red because of accumulated porphyrins (uroporphyrin), anemia; due to photodynamic action on porphyrins converting them to toxic molecules
Combination of acute and cutaneous porphyria: both neurological and skin symptoms
Autosomal dominant, so enzyme activity 50% reduced
Secondary (acquired) porphyria
Chemical, toxic substances (lead) that inhibits heme synthesis or induces ALA synthetase
Why is it bad to have too much iron (Fe2+)?
Toxic free radical (hydroxyl) is created if too uch Fe2+ around
Where is most of the iron in the body found?
RBCs (“functional” compartment)
How much of daily iron need (20-25mg) is recycled?
90% of daily iron need recycled from macrophages that engulfed senescent RBCs
How do we get rid of iron?
No specific way to get rid of iron!
Bleeding, menstruation, sloughed mucosal cells
Transferrin
Binds 2 molecules of Fe3+ in plasma
Protects against free radical generation by free Fe
Transferrin-Fe complex endocytosed into cells by binding transferrin receptors TfR1 or TfR2
Absorption of dietary Fe by duodenum
1) Fe3+ converted to Fe2+ by ferrireductase on membrane
2) Fe2+ in lumen through DMT1 into duodenal enterocyte; Heme through HCP1 into duodenal enterocyte
3) Fe2+ back to Fe3+ and binds apoferritin to form FERRITIN (huge protein that binds 4500 atoms of Fe3+)
4) Fe2+ transported into plasma by membrane protein ferroportin
5) Once in plasma, Fe2+ converted to Fe3+ again by ferroxidase
6) Fe3+ in plasma must bind transferrin
What do plasma levels of ferritin tell us?
Plasma level of ferritin tell us cell level of ferratin (a SMALL amount of ferritin leaks into plasma), and cell level of ferratin tells us person’s Fe level
(Except in inflammatory disorder, liver disease, cancer–then ferritin level can be normal/high but person has really low level of Fe)
What happens to apoferritin synthesis when plasma Fe (ie intracellular levels of Fe) is low?
Apoferritin synthesis inhibited
What happens to Fe3+ in erythroblasts (RBC precursors in stem cells) and other cells?
Fe3+ bound within ferratin
Fe3+ reduced to Fe2+ by ferrochelatase and put into heme
Distribution of Fe in the body
Storage compartment: bone marrow, liver cells, reticulo-endothelial macrophages
Transport compartment: serum transferrin
Functional compartment: hemoglobin, myoglobin, cytochromes, non-heme iron proteins
Causes of anemia (low RBCs or low hemoglobin) due to abnormal Fe metabolism
1) Fe deficiency
2) Defective Fe storage
3) Defective Fe utilization in erythroblasts
Anemia due to Fe deficiency (most common anemia in US)
1) Increased physiological demand (growing adolescent)
2) Inadequate intake (diet or absorption)
3) Chronic blood loss
Why are females more susceptible to iron deficiency anemia?
Females lose 1mg/day like men but additional 0.5-1mg/day because of menstruation
Pregnant women require more (2mg/day) for fetal needs, placenta, and expanded maternal blood volume
Anemia due to defective Fe storage
1) Defect secondary to chronic disease (TB; this is second most common anemia in the US)
2) Due to entrapment of Fe in macrophages (results in low plasma Fe, high intracellular/plasma ferritin)
How is hepcidin involved in anemia due to defective Fe storage
Hepcidin is liver peptide that binds/internalizes ferroportin in macrophages and duodenal enterocytes so now Fe has no way out of the cell
Hepcidin induced by cytokines (so in chronic disease, get low Fe levels in blood but high plasma/intracellular ferritin levels)
Note: giving iron doesn’t help the problem
How is iron transported from mother to fetus?
Ferroportin brings Fe from mother to fetus across placenta
Fetal hepcidin is present to ensure not too much Fe transported to fetus
Anemia due to defective Fe utilization in erythroblasts (in bone marrow)
Called sideroblastic anemia b/c Fe accumulation (as ferritin) in erythroblasts then called sideroblasts (excess ferritin can precipitate to form hemosiderin)
Can be hereditary (X linked) defect in heme synthesis or acquired defect (lead poisoning leading to inhibition of heme synthesis enzymes)
Note: heme made in erythroblasts of bone marrow (and in liver), and here heme not made correctly
Adult-onset hereditary hemochromatosis
Too much iron in body (absorbed from GI tract)
Decreased hepcidin activity (because of defect in HFE) causes increased Fe absorption from duodenum across ferroportin –> high plasma Fe levels and Fe accumulation in tissues
Clinical effects: bronze skin, liver/heart/pancreas damage, manifests in adults
HFE (HLA-linked Fe-loading gene): stimulates hepcidin synthesis
Common in Northern Europeans but incomplete penetrance (also depends on diet, etc)
Juvenile onset hereditary hemochromatosis
Too much iron in body (absorbed from GI tract)
Due to mutation in hepcidin or another gene involved in Fe metabolism
Manifest early in life and are highly penetrant
Hemostasis
A process that minimizes hemorrhage when a blood vessel wall is damaged and that minimizes formation of untimely clots (thrombi) in undamaged vessels
Primary hemostasis
1) Vasoconstriction of damaged vessels (only impt in uterus)
2) Formation of temporary seal by platelets
Secondary hemostasis
Formation of a blood clot/fibrin clot (coagulation)
Formation of platelet plug/seal (primary hemostasis)
1) Damage to blood vessel endothelium
2) Platelets contact damaged vessel endothelium
3) Platelets change shape to interact with other platelets better and adhere to subendothelium better (using von Willebrand factor in endothelium, etc)
4) Platelets also release ADP and thromboxane A2 (by exocytosis), which bind to other platelets and trigger their activation
5) Note: remember platelets need to use von Willebrand factor (vWf; INSIDE endothelial cells) to bind to vessel endothelium
6) Platelets accumulate to entirely fill gap by interacting with other platelets: the plasma protein fibrinogen acts as a crossbridge between platelets which all have GP IIb/IIIa receptors. Now we have formed the platelet plug/seal
(Next, platelet plug/seal becomes stabilized by formation of blood/fibrin clot (coagulation))
Note: activated platelets also release certain growth factors that facilitate healing of injured blood vessels
What are two ways platelets can get activated?
1) Bind damaged endothelium (triggered by tissue factor then thrombin cleaves thrombin receptor on platelets to activate them)
2) Activated by binding ADP or TxA2
What prevents clotting from happening when there is no endothelium damage?
Prostacyclin (PGI2) released by normal endothelial cells and surface integrity inhibit platelet activation/aggregation
Plavix (clopidogrel)
Inhibits hemostasis by blocking platelet activation
Inhibits ADP binding to receptor on platelets
Aspirin
Inhibits hemostasis by blocking platelet activation
COX blocker: blocks synthesis of TxA2 by blocking COX1 in platelets
How does fibrinogen contribue to platelet interaction?
Figrinogen helps platelets interact by acting as a crossbridge between 2 platelets
Fibrinogen binds GP IIb/IIIa receptors (complex of glycoproteins IIb and IIIa) on platelets
Abciximab
Monoclonal antibody that binds GPIIb/IIIa to block those sites so fibrinogen can’t bind
Is used to decrease risk of thrombus formation
Formation of blood clot (secondary hemostasis)
This happens after platelet plug of fibinogen holding platelets together has happened
Intrinsic pathway: damaged surface –> kininogen and kallikrein recognize damage and activate XII (Hageman factor) –> activate XI –> activate IX –> VIIIa bound to IXa activates X –> final common pathway
Extrinsic pathway: trauma –> activate VII –> tissue factor bound to VIIa activates X –> final common pathway
Final common pathway: Va with Xa converts prothrombin to thrombin –> thrombin converts fibrinogen to fibrin –> XIIIa cross-links fibrin to clot
Soft clot vs. hard clot
Soft clot: 3D aggregate of fibrin monomers with no crosslinks
Hard clot: covalent bonds between gln and lys resides of adjacent fibrin monomers (crosslinks) created by Factor XIIIa
Requirements of blood clotting
1) Rapid and efficient
2) No clotting under normal conditions
3) Self-limiting
4) Removal of clots as healing occurs
Why don’t fibrin molecules aggregate when no damage?
No fibrin molecules present–only fibrinogen!
Fibrinogen is a dimer and each monomer has (A-alpha, B-beta, gamma) peptides and does not aggregate because A and B are negatively charged and repel each other
Thrombin (only activated from prothrombin by clotting cascade) converts fibrinogen to fibrin, by removing negatively charged A and B to create (alpha, beta, gamma) peptides.
Now Factor XIIIa helps fibrin monomers aggregate
If you take blood out of the body and put it in a test tube, will it clot?
Yes, kininogen and kallikrein recognize test tube as damaged blood vessels and initiate intrinsic pathway
Tissue factor
Intra-membranous protein in connective tissue of vessel wall that sticks out from fibroblast when there is trauma/damage
In vivo, this is what triggers clotting cascade!
Binds to VIIa to activate it (so VIIa can turn X –> Xa)
Exists in blood in inactive state on particles derived from dead WBCs, and activation occurs by unknown mechanism (maybe lipid reorganization)
Is there crosstalk between intrinsic and extrinsic pathway?
In vivo, yes crosstalk (factor VIIa can activate factor IX)
In vitro, no crosstalk (with no tissue factor you completely eliminate extrinsic factor)
If tissue factor initiates clotting cascade, why do we need the intrinsic pathway?
Extrinsic pathway (using VIIa) initiates clotting and becomes active, but then inhibitors come into play to inhibit extrinsic pathway (inhibit VIIa)
Intrinsic pathway is needed to prolong clotting cascade as extrinsic pathway becomes inhibited
Clotting initiated by extrinsic pathway, completed/prolonged by intrinsic pathway
Why distinguish between intrinsic and extrinsic pathways?
Can use different clotting tests to determine where deficiency is
What happens if you’re deficient in kininogen, kallikrein, or Factor XII?
No problem! Can still clot because of tissue factor in vivo and extrinsic pathway
Do you need platelets for clotting?
NO!
Platelets enhance clotting in vivo by assembling activated clotting factors on their surface, but are not required
Recombinant factor VIIa
Used to speed clotting in people with hemorrhage, etc
Classes of factors
Serine proteases: remove part of preursor portion (II, VII, IX, X, XI, XII, kallikrein)
Nonprotease activators/accelerators: stabilize other factors (V, VIII, tissue factor, high molecular weight kininogen)
Stabilizer of factor VIII: von Willebrand factor (in plasma)
Transamidase that crosslinks fibrin: factor XIII
Nonprotein factors: stabilize many clotting factors (Ca2+ and membrane phospholipids)
Roles of thrombin
1) Cleaves thrombin receptor on platelets to activate platelets (second pathway of platelet activation, which is initiated by tissue factor)
2) Cleaves V and VIII to form active Va and VIIIa
3) Limits clot formation by eventually cleaving Va and VIIIa (along with thrombomodulin)
How do we limit clot formation?
1) Thrombin binds thrombomodulin (intramembranous protein that sticks out into blood) –> thrombin-thrombomodulin complex activates protein C –> active protein C binds protein S –> together, cleave Va and VIIIa –> clotting is terminated (hydrolysis products)
2) Antithrombin (produced by liver) binds active protease (IIa (thrombin) and Xa especially) and inactivates them –> antithrombin-inactive protease complex (heparin binds this complex and increases activity 1000x)
Heparin
Anti-coagulant, injected IV
Prevents clot formation by increasing antithrombin activity
Which clotting factors require binding of Ca2+, and thus require vitamin K?
II, VII, IX, X
Protein C, Protein S
How does vitamin K act on clotting factors to help activate them?
Vitamin K modifies clotting factors by turning Glu-to-Gla residues (just add carboxy group) so that they can bind Ca2+ and become active
Vitamin K does this modification in the liver, so once blood is sent out from the liver, it has glu-to-gla-transformed (ready to be active) clotting factors
Warfarin (coumadin)
Anti-coagulant, prevents clot formation–rat poison causes them to bleed to death
Inhibits action of vitamin K (vitamin K needs to be reduced to work again, and warfarin inhibits this)
Warfarin inhibits vitamin K reductase
Activity of warfarin function of age, race, weight, height, smoking, other meds, diet, and >30 genes
Warfarin acts in the liver!!
Warfarin competes with oxidized vitamin K, so if OD on warfarin, can give vitamin K to try to stop hemorrhage but will take a long time
Activity of vitamin K reductase and warfarin
1) Vitamin K oxidized when it does glu-to-gla to activate clotting factors
2) Vitamin K reductase reduces vitamin K back so it can activate more clotting factors
Warfarin inhibits vitamin K reductase
Warfarin inactivated by CYPs
If you add warfarin or heparin to test tube with blood, will blood clot?
Heparin: blood will not clot if heparin in test tube because heparin combines with antithrombin and inhibits proteases/clotting factors from forming clots
Warfarin: blood WILL clot if warfarin in test tube because clotting factors in blood have already done glu-to-gla transformation (in liver) and are active
Low molecular weight heparin (LMWH)
MW </= 6,000
1) Less non-specific binding to plasma proteins, so get better control of true dosage
2) Fewer adverse side effects than heparin (inhibition of platelets)
3) LMWH can be injected subcutaneously so can be administered at home
Fondaparinux
Synthetic pentasaccharide similar to heparin but more specific for Factor Xa
Oxalate, EDTA
Ca2+ chelators; bind up Ca2+ so can’t activate clotting factors and clotting can’t occur
If add these to test tube, blood won’t clot
Never use these in vivo of course!
Synthetic protease inhibitors (anticoagulants) that don’t require monitoring of patient’s clotting activity
Dabigatran (Pradax): specific inhibitor of thrombin
Rivaroxaban and Apixaban: specific inhibitor of factor Xa
Bivalirudin: synthetic peptide analog of hirudin, thrombin inhibitor present in saliva of leech
How do you tell if OD was of heparin or warfarin?
Mix normal blood with patient’s blood:
If heparin OD then normal blood’s clotting factors will be cleaved by heparin and combined blood won’t clot
If warfarin OD, then normal blood’s clotting factors have undergone glu-to-gla transformation and will help combined blood clot (basically can restore clotting function with normal clotting factors from normal blood)
“Heparin just freaking kills everything in its way”
After a clot is formed, how do we get rid of that clot as healing occurs?
1) Plasminogen from liver converted to plasmin by TPA (from vascular endothelium) –> plasmin breaks down fibrin clot and releases fibrin split products (D-dimers)
Recombinant forms of TPA used in stroke patients: Alteplase and Tenecteplase
D-dimer
D domains on fibrin monomers attach to each other to form dimers when plasmin breaks down a fibrin clot–called D-dimers
Antibody can recognize D-dimers but not fibrin monomers
D-dimers elevated in thromboembolism in brain, infection, neoplasia
Bleeding time
Lab test (not used anymore) to assess primary hemostasis
Incision in wrist and see how long takes to stop bleeding
Prothrombin time (PT)
Clotting time in presence of added tissue factor (obtained from animal brain tissue)
Measures extrinsic and final common pathway
International normalized ratio (INR) = PT of patient’s blood:PT of normal blood
(INR of normal blood is 1)
Activated partial thromboplastin time (APTT or PTT)
Add kaolin (clay) which triggers intrinsic pathway and see how long until clotting
Measures intrinsic and final common pathways
Activated clotting time (ACT)
Add diatamaceous earth to blood to see time to clotting
Similar to APTT
Used to monitor heparin levels when high doses given (in cardiac bypass surgery)
Russell’s Viper Venom Test
Russell’s viper venom (converts factor X to Xa) added to blood in test tube and time to clotting measured
Which tests are used to assess warfarin and heparin levels?
PT used for warfarin levels (remember PT extrinsic, warfarin given outpatient/oral)
PTT used for heparin levels (remember PTT intrinsic, heparin given inpatient/IV)
(PT and PTT should be affected by both but aren’t actually in practice)
Anti-Xa activity test used for LMWH and Fondaparinux (PTT not sensitive to these drugs)
Potential causes of bleeding disorder
1) Vasoconstriction problem (only women)
2) Thrombocytopenia (decreased platelets)
3) Platelet dysfunction
4) Deficiency/defect in in clotting factor (hereditary hemophelia vs. acquired vitamin K deficiency)
5) Inhibitors of clotting factors (heparin, antibodies if autoimmune)
6) Excessive fibrinolysis
7) Combo of above
What is wrong with babies a lot of the time that affects blood clotting?
Many babies vitamin K deficient at birth, so always administer vitamin K at birth
Vitamin K made by bacteria in GI tract but baby doesn’t have bacteria in GI tract at birth
Mom can pass on some vitamin K to baby but baby doesn’t have fat to store vitamin K in
Baby’s liver not fully functional at birth so needs extra vitamin K to do glu-to-gla transformation
Hemophilia A
Hereditary blood clotting disease (can bleed easily, bruise, bleed into joints)
X-linked recessive deficiency in factor VIII (intrinsic pathway, measure PT!)
Have <5% normal factor VIII activity
(A = 8)
Hemophilia B (Christmas Disease)
Hereditary blood clotting disease (can bleed easily)
X linked recessive deficiency of factor IX (intrinsic pathway, measure PT!)
(B = 9)
von Willibrand Disease
Hereditary blood clotting disease (can bleed easily)
Deficiency of von Willibrand factor
Fairly common
Thrombophilia
Love to clot
Hypercoagulation disorders
Untimely intravascular clot formation due to imbalance between anticoagulant and prothrombotic activities of plasma
Can be hereditary or acquired
Hereditary thrombophilia
1) Factor V mutation (Factor V Leiden): mutation makes factor V resistant to protein C so can’t break down clot, keep on clotting
2) Prothrombin G20210A mutation: overproduction of prothrombin
3) Deficiency in level or activity of protein C, protein S, antithrombin
Note: mutations above only manifest when other factors occur also (oral steroid contraceptives cause increases/decreases in clotting factors) or when have another thrombophilia mutation too
Acquired thrombophilia
Caused by:
1) Vascular infection or inflammation (induced expression of tissue factor; have ongoing coagulation/inflammation that decreases levels of antithrombin, protein C, protein S)
2) Severe trauma
3) Cancer (induced expression of tissue factor)
4) Pregnancy (hormone changes)
5) Antiphospholipid syndrome: autoimmune antibodies against phospholipids on cell membranes cause increased clots (can also cause fetal loss in pregnancy)
Treatment: anticoagulants or platelets or plasma, control underlying condition (ex: treat infection)
Disseminated intravascular coagulation (DIC)
Widespread, occurs throughout most of vasculature; get both thrombi and bleeding
Systemic activation of coagulation –> intravascular deposition of fibrin –> thrombosis of small/midsize vessels can cause organ failure –> increased clotting means depletion of platelets and coagulation factors –> bleeding
Medical emergency
Neonatal Hemochromatosis
Rare, severe liver disease due to increased iron/hemosiderin, and iron/hemosiderin deposition (hemosiderosis) on liver and other tissues
Clinical features: fetal death or illness/impaired growth at birth, evidence of liver failure (low albumin, low glucose, coagulopathy, low fibrinogen, thrombocytopenia, anemia), siderosis
Diagnose by looking for siderosis in biopsied liver or salivary gland
Pathogenesis: maternal IgG against fetal liver tissue damage baby’s liver –> damaged liver can’t produce enough hepcidin –> too much iron transport across placenta –> iron deposits on liver and other tissue
(Note: this mother will probably have the same thing happen in future pregnancies)
Tx: anti-oxidents, liver transplantation; during pregnancy weekly IV administration to mother of IgG from other donors to compete with mother’s anti-fetal liver IgG so donor IgGs cross placenta more than mother’s bad IgG
How are warfarin and heparin used in the clinic?
Heparin: IV; in-patient
Warfarin: oral; outpatient (even though easy to over- or under-dose warfarin!)
Anti-platelet agents
Help prevent formation of initial platelet plug
1) Aspirin (NSAIDs)
2) Plavix (Clopidogrel)
3) Abciximab
Anticoagulants
Help prevent full formation of blood clot (after platelet plug has formed)
1) Ca2+ chelators (oxalate, EDTA)
2) Heparin (unfractionated heparin, LMWH (enoxaparin = Lovenox), Fondaparinux (more specific for factor Xa)
3) Warfarin (= Coumadin)
4) Synthetic specific protease inhibitors that don’t require patient monitoring (Dabigatran (Pradax) and Bivalirudin inhibit thrombin; Rivaroxaban and Apixaban inhibit Xa)
Recombinant forms of TPA used to treat acute ischemic stroke
Alteplase
Tenecteplase
