Module 5 Flashcards
1
Q
- are complex chemical compounds that are large heterocyclic organic ring structures
- are composed of four modified pyrrole (5- membered organic ring) subunits connected by methine (=CH-) bridges
A
Porphyrins
2
Q
- The naturally occurring porphyrins of biological significance are the hemes
- serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen
- Synthesized in most organisms via a highly conserved biosynthetic route
- Produced in virtually all mammalian tissues
A
Heme
3
Q
- Cofactor
- Consists of a porphyrin (protoporphyrin IX)
- Iron (Fe++) chelate in the center
- Conjugated (alternating) double bonds absorb light»_space; a color
- A tightly bound prosthetic group of hemoglobin, myoglobin, the cytochromes, and other proteins
- Bound to its apoproteins
– Noncovalently: HEMOGLOBIN, MYOGLOBIN
– Covalently: CYTOCHROME C
A
Heme
4
Q
Heme-containing proteins
A
• Hemoglobin • Myoglobin • Enzymes – Catalases – Peroxidases – Tryptophan pyrrolase – Prostaglandin synthase – Guanylate cyclase – NO synthase – Mitochondrial cytochromes
5
Q
Sources of heme:
A
– Endogenous synthesis
• Bone marrow
• Liver
– Intestinal absorption of dietary heme
6
Q
Synthesis sites
A
• Marrow
– 70-80% heme synthesis, mostly in erythroblasts
and proerythroblasts
• Liver
– second most important site of heme synthesis
– High content of cytochrome p450
7
Q
Heme biosynthesis
A
• The heme biosynthetic pathway and its subcellular compartmentation are probably identical in all mammalian cells
• 8 enzymes: 4 cytoplasmic, 4 mitochondrial
• Compartmentalized
– Initial and last three enzymes: mitochondrial
– Intermediate steps: cytosol
– Significance? Heme regulates ALA synthase (rate limiting step)
8
Q
Three divisions of heme biosynthesis
A
- Formation of the precursor molecule ALA
- Formation of the first cyclic tetrapyrrole uroporphyrinogen III
- Conversion of uroporphyrinogen III into heme
9
Q
Formation of the precursor molecule ALA (Aminolevulinic Acid)
A
- First and rate-limiting reaction: condensation of glycine and succinyl-CoA to 5-aminolevulinic acid (ALA)
- ALA represents the sole source of carbon and nitrogen atoms necessary for heme formation
10
Q
Formation of the precursor molecule ALA
A
• Catalyzed by two different ALA synthases
(cofactor: PYRIDOXAL 5-PHOSPHATE)
– expressed ubiquitously (ALAS1)
– expressed only in erythroid precursors (ALAS2)
11
Q
- The next four biosynthetic steps take place in the cytosol
- ALA dehydratase (ALAD) converts two molecules of ALA to a monopyrrol porphobilinogen (PBG)
- Two subsequent enzymatic steps convert four molecules of PBG into the cyclic tetrapyrrole uroporphyrinogen III, which is then decarboxylated to form coproporphyrinogen III
A
Formation of the first cyclic tetrapyrrole uroporphyrinogen III
12
Q
The fate of ALA
A
• Following its synthesis, ALA exits the mitochondria by an unknown mechanism
• Once in the cytosol, two molecules of ALA form the monopyrrole porphobilinogen by a condensation reaction catalyzed by aminolevulinate dehydratase (ALAD)
(cofactor: zinc)
13
Q
The next step in the pathway involves the head-to-tail
condensation of four molecules of porphobilinogen to produce the linear tetrapyrrole intermediate, __.
A
hydroxymethylbilane
14
Q
The most important fate of __ is the regulated, enzymatic conversion to uroporphyrinogen III, the next intermediate on the path to heme
A
hydroxymethylbilane
15
Q
- catalyzes the terminal step of heme biosynthesis, namely the insertion of ferrous iron into protoporphyrin IX
A
Ferrochelatase
16
Q
Heme biosynthesis in a nutshell
A
• The first step occurs in the mitochondria and involves the condensation of succinyl CoA and glycine to form 5-aminolevulinic acid (ALA), catalyzed by ALA synthase
(ALA-S)
• The next four biosynthetic steps take place in the cytosol. ALA dehydratase (ALA-D) converts two molecules of ALA to a monopyrrol porphobilinogen (PBG). Two subsequent enzymatic steps convert four molecules of PBG into the cyclic tetrapyrrole uroporphyrinogen III, which is then decarboxylated to form coproporphyrinogen III
• The final three steps of the biosynthetic pathway,
including the insertion of ferrous iron into protoporphyrin
IX by ferrochelatase, occur in the mitochondria
17
Q
Type: X-linked sideroblastic anemia (erythropoietic)
Major signs and symptoms: Anemia
Lab Test Result: Red cell counts and hemoglobin
decreased
A
ALA synthase 2 (ALAS2)
18
Q
Type: ALA dehydratase deficiency (hepatic)
Major signs and symptoms: Abdominal pain, neuropsychiatric symptoms
Lab Test Result: Urinary ALA and coproporphyrin III increased
A
ALA dehydratase
19
Q
Type: Acute intermittent porphyria (hepatic)
Major signs and symptoms: Abdominal pain, neuropsychiatric symptoms
Lab Test Result: Urinary ALA and PBG increased
A
Uroporphyrinogen I synthase
20
Q
Type: Congenital erythropoietic (erythropoietic)
Major signs and symptoms: Photosensitivity Urinary, fecal, and red cell
Lab test Result: uroporphyrin I increased
A
Uroporphyrinogen III synthase
21
Q
Type: Porphyria cutanea tarda (hepatic)
Major s/sx: Photosensitivity
Lab Test Result: Urinary uroporphyrin I increased
A
Uroporphyrinogen decarboxylase
22
Q
Type: Hereditary coproporphyria (hepatic)
Major s/sx: Photosensitivity, abdominal pain, neuropsychiatric symptoms
Lab Test Result: Urinary ALA, PBG, and coproporphyrin III and fecal coproporphyrin III increased
A
Coproporphyrinogen oxidase
23
Q
Type: Variegate porphyria (hepatic)
Major s/sx: Photosensitivity, abdominal pain, neuropsychiatric symptoms
Lab Test Result: Urinary ALA, PBG, and coproporphyrin III and fecal protoporphyrin IX increased
A
Protoporphyrinogen oxidase
24
Q
Type: Protoporphyria (erythropoietic)
Major s/sx: Photosensitivity
Lab Test Result: Fecal and red cell protoporphyrin IX increased
A
Ferrochelatase
25
PORPHYRINS VS. PORPHYRINOGENS
PORPHYRINOGENS are colorless, whereas the various porphyrins are all colored
The double bonds joining the pyrrole rings in the PORPHYRINS are responsible for their fluorescence
Spectrophotometry is used to test for porphyrins and their precursors (for diagnosis of porphyrias)
26
- Group of disorders due to abnormalities in the pathway of biosynthesis of heme
- Genetic or acquired
- If the enzyme lesion occurs early in the pathway prior to formation of porphyrinogens, ALA and PBG accumulates in body tissues causing abdominal pain and neuropsychiatric symptoms
- Later blocks cause photosensitivity
PORPHYRIAS
27
Enzyme affected: Ferrocheletase, ALA dehydrate
| Accumulated substrate in Urine: Coproporphyrin, ALA
Lead poisoning
28
Affected Enzyme: Uroporphyrinogen I synthase
| Accumulated Substrate in Urine: Porphobilinogen, δ-ALA
Acute intermittent porphyria
29
Affected Enzyme: Uroporphyrinogen decarboxylase
| Accumulated substrate in Urine: Uroporphyrin
Porphyria cutanea tarda
30
- Treatment is symptomatic
- Ingestion of large amounts of carbohydrates or administration of hematin may repress ALAS 1 resulting in diminished production of harmful heme precursors
- Patients experiencing photosensitivity may benefit from administration of beta-carotene, aside from administering sunscreens
PORPHYRIAS
31
Biological importance of heme
• Heme is a ubiquitous molecule that is involved in many essential biological processes, including oxygen transport,
respiration, photosynthesis, drug detoxification and signal transduction
32
Why regulate heme?
* Free heme is a potent pro-oxidant, leading to the formation of reactive oxygen species that can damage a variety of biological molecules
* Heme can associate with phospholipid membranes, altering bilayer structure and, thus, causing cell disruption
* For this reason, the cells strictly regulate heme homeostasis
33
(Heme catabolism)
• As the heme is not recycled, most cells containing heme proteins have the microsomal mixed function oxygenase, heme oxygenase, which enzymatically degrades heme to __
biliverdin, carbon monoxide, and iron
34
Effects of heme oxygenase activity
* In mammals, CO, a gaseous messenger, has anti- inflammatory and anti-apoptotic effects
* Biliverdin and its reduced product bilirubin may function as important antioxidants
35
Heme oxygenase
* Humans harbor two distinct heme oxygenase genes identified as HMOX1 and HMOX2
* The heme oxygenase enzyme encoded by the HMOX1 GENE is the rate-limiting enzyme of heme catabolism
* Both HMOX1 and HMOX2 genes are constitutively expressed, however, the activity of the HMOX1 encoded enzyme is inducible by heme, heavy metals, and conditions of stresssuch as hypoxia
36
* The red cell with the largest pool of heme protein, hemoglobin, contains no heme oxygenase
* Enzymatic degradation of the red cell heme occurs only after the senescent red cells are removed by the reticuloendothelial system
Heme catabolism
37
• The largest repository of heme in the human body is in RBCs which have a life span of about 120 days
• There is a turnover of about 6 g/day of hemoglobin, which presents 2 problems:
– The porphyrin ring is hydrophobic and must be
solubilized to be excreted
– Iron must be conserved for new heme synthesis
Heme catabolism
38
Sources of heme catabolites
* Roughly 80% of heme destined for degradation and excretion comes from senescent erythrocytes
* 20% comes from premature erythrocytes in the bone marrow which are destroyed prior to release into the circulation and a minor component is derived from other cell types
39
Heme breakdown to bilirubin
• Within hepatic and splenic macrophages,heme is first converted to bilirubin in a two-step enzymatic process which employs biliverdin as an intermediate (enzyme:
BILIVERDIN REDUCTASE)
40
Oxidative metabolism of heme by __, giving rise to CO, iron, biliverdin, and bilirubin
HO and biliverdin reductase
41
Heme breakdown to bilirubin
* These steps result in oxidation and opening of the heme ring
* Macrophages then excrete the resultant bilirubin into the plasma as unconjugated bilirubin
42
Biliverdin reductase
* There are two biliverdin reductase genes in humans identified as BLVRA and BLVRB
* The enzyme encoded by the BLVRA gene is pricipally responsible for the catabolism of biliverdin
* The enzyme encoded by the BLVRB gene catalyzes the reduction of not only biliverdin but also a variety of flavins, such as riboflavin, FAD or FMN, and methemoglobin -> aka NADPHdependent flavin reductase
43
Fate of bilirubin
* Bilirubin is significantly less extensively conjugated than biliverdin causing a change in the color of the molecule from blue-green (biliverdin) to yellow-red (bilirubin)
* Peripherally arising bilirubin is transported to the liver in association with albumin, where the remaining catabolic reactions take place
44
* Within the hepatocyte, the enzyme UGT1A1 (UDP-glucuronyltransferase) covalently attaches one or two molecules of glucuronic acid to bilirubin, generating either bilirubin mono- or di-glucuronide
* These glucuronic acid-attached species of bilirubin are termed CONJUGATED BILIRUBIN
* The increased water solubility of the tetrapyrrole facilitates its excretion with the remainder of the bile as the bile pigments
Bilirubin conjugation
45
* The most important of conjugation reactions
* Reaction does not proceed spontaneously
* Requires the activated form of glucuronic acid -> glucuronic acid uridine diphosphate
Glucuronidation
46
- occurs when bilirubin in the blood exceeds 1 mg/dL (17.1 mol/L)
- due to excess bilirubin production or liver failure
- Obstruction of the excretory ducts of the liver causes hyperbilirubinemia
- When bilirubin reaches 2-2.5 mg/dL in blood, it diffuses into tissues causing JAUNDICE
Hyperbilirubinemia
47
TYPES OF HYPERBILIRUBINEMIA
1. Retention hyperbilirubinemia due to overproduction of bilirubin (UNCONJUGATED BILIRUBIN)
2. Regurgitation hyperbilirubinemia due to the reflux into the bloodstream secondary to biliary obstruction (CONJUGATED BILIRUBIN)
48
- Because of its hydrophobicity, only unconjugated bilirubin can cross the blood-brain barrier into the CNS
- Encephalopathy due to hyperbilirubinemia can occur only in connection with unconjugated bilirubin, as found in retention hyperbilirubinemia
Kernicterus
49
Jaundice
- Conjugated bilirubin can appear in urine
- CHOLURIC JAUNDICE occurs only in regurgitation hyperbilirubinemia
- ACHOLURIC JAUNDICE OCCURS only in the presence of an excess of unconjugated bilirubin
50
UNCONJUGATED HYPERBILIRUBINEMIA
Crigler-Najjar syndrome type I
- Congenital nonhemolytic jaundice
- Autosomal recessive
- Due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues
Crigler-Najjar syndrome type II
- Like type I but more benign
51
UNCONJUGATED HYPERBILIRUBINEMIA
Gilbert syndrome
- Also has mutations in genes encoding bilirubin-UGT but retains 30% of enzyme activity
Toxic hyperbilirubinemias
- Chloroform, carbon tetrachloride, acetaminophen, viral hepatitis, cirrhosis, Amanita mushroom
52
CONJUGATED HYPERBILIRUBINEMIA
1. Biliary Obstruction
- Regurgitation into hepatic veins and lymphatics
- Conjugated bile in blood and urine
2. Dubin-Johnson syndrome
- Benign autosomal recessive
- Mutation in the gene encoding MRP-2 for the secretion of conjugated bili into bile
3. Rotor syndrome
- Chronic conjugated hyperbilirubinemia and normal liver
53
Major functions of Blood
- Regulation of body temperature by the distribution of body heat
- Defense against infection by the white blood cells and circulating antibodies
- Transport of hormones and regulation of metabolism
- Transport of metabolites
- Coagulation
54
ALL BLOOD CELLS ARE DERIVED FROM HEMATOPOIETIC STEM CELLS
Proerythroblast- reticulocyte – erythrocyte
Myeloblast – baso, eosi, neutrophil
Monoblast – monocyte (macrophage)
Megakaryoblast – platelet
Lymphoblast – t cell ( active t cell), b cell (plasma cell)
55
Function
- Delivery of oxygen to tissues
- Disposal of CO2 and protons from tissues
Red Blood Cells
56
Structure of RBC
- Composed of membrane surrounding a solution of hemoglobin
- Membrane has the Chloride-Bicarbonate antiport mechanism which allows CO2 transport from the periphery to the lungs
- Anucleated
- No intracellular organelles
- Biconcave shape
- Increases surface area to volume ratio facilitate gas exchange
57
- New RBC in the circulation which still contain ribosomes and elements of endoplasmic reticulum
- 1% of total RBC count
- Increased in hemolytic anemias
RETICULOCYTES
58
- Major regulator of human erythropoiesis
| - Glycoprotein, synthesized in the kidneys in response to hypoxia
ERYTHROPOIETIN
59
Erythropoietin interacts with RBC progenitors via specific receptors
1. Burst-forming unit-erythroid (BFU-E)
| 2. Colony-forming unit-erythroid (CFU-E)
60
Other hematopoietic growth factors
Granulocyte-colony stimulating factor (G-CSF) – for granulocytes
Granulocyte-Macrophage colony stimulating factor (GM-CSF) – for granulocyte and macrophages
61
RBC METABOLISM
- RBC has no mitochondria
- ATP produced only by glycolysis
* Source of energy: Glucose (90% anaerobically degraded to lactate, 10% by HMP shunt)
* Entry of glucose is by FACILITATED DIFFUSION THROUGH glucose transporter (GLUT1) which is not insulin-dependent
- 2, 3 BPG, a side product of glycolysis, is important in the unloading of oxygen by hemoglobin
- NADPH from PPP is needed by RBC in the formation of GSH which is required in the degradation of H2O2
62
Reactive oxygen species (ROS) can react with protein, nucleic acid, lipids and other molecules to alter their structure and produce tissue damage
OXIDATIVE STRESS
63
- RBC generates powerful Reactive Oxygen Species (ROS) during metabolism
```
- These oxidants are not only produced in RBC but also in other cells in the body
Superoxide (O2 • -)
Hydrogen peroxide (H2O2)
Peroxyl radicals (ROO •)
Hydroxyl radicals (OH •)
```
RBC METABOLISM
64
(RBC metabolism)
- Reaction in pentose phosphate pathway
- Deficiency can result in hemolytic anemia or intake of certain drugs (primaquin, sulfonamides) or chemicals
(napthalene) due to generation of H2O2
- rate limiting enzyme in the oxidative phase of PPP which generates NADPH – keeps gluta reduced and detoxifies free radicals and peroxides
Glucose 6-phosphate Dehydrogenase
65
RBC metabolism: Accumulation of H2O2
1. Peroxidation of lipids in membrane -> lysis of RBC
2. Oxidation of –SH groups in hemoglobin -> precipitation of proteins inside RBC -> formation of Heinz bodies (indicative of oxidative stress)
66
RBC Metabolism: Formation of Methemoglobin
- Results from the oxidation of Fe++ to Fe+++ in hemoglobin
- Cannot transport oxygen
- Can be reduced back to Fe++ by NADH-Cytochrome b5 Methemoglobin Reductase
67
METHEMOGLOBINEMIA
1. Inherited - Deficiency of methemoglobin reductase; Presence of Hb M (substitution of His to Tyr)
2. Acquired - Ingestion of certain drugs (sulfonamides) and chemicals (aniline)
* Cyanosis evident when over 10% of Hb is in the Met form
68
- Bilayer composed of ~50% lipids and 50% protein
- Lipids are phospholipids and cholesterol (Important in membrane fluidity)
- Proteins are mostly glycoproteins (Named according to their migration in SDS-PAGE)
RBC MEMBRANE
69
(RBC membrane proteins)
- Anion exchange protein (band 3)
- Glycophorin A, B, and C
Integral membrane proteins
70
(RBC membrane proteins)
- Spectrin (band 1 and 2)
- Ankyrin (band 2.1)
- Protein 4.1
- Actin (band 5)
Peripheral membrane proteins
71
- Transmembrane glycoprotein with its C-terminal on external surface of membrane and N-terminal on cytoplasmic surface
- Multipass membrane protein extending across the bilayer at least 10 x
- Forms a tunnel permitting the exchange of chloride for bicarbonate
- N-terminal binds other protein – Hb, protein 4.1, 4.2, ankyrin and several glycolytic enzymes
Anion exchange protein
72
- 131 aa, heavily glycosylated (~60%)
- N-TERMINAL CONTAINS 16 oligosaccharide chain containing SIALIC ACID located on RBC surface
- C-TERMINAL extends to cytosol and binds protein 4.1 which in turn binds SPECTRIN
Glycophorin
73
- Major protein of the cytoskeleton
- Composed of 2 polypeptides – α and β which aligns in anti-parallel manner and loosely intertwined
- Folds into triple stranded α-helical coils joined by non-helical segment; interact head to head to form tetramers
Spectrin
74
- Binds spectrin and band 3
| - Sensitive to proteolysis accounting for appearance of bands 2.2, 2.3 and 2.6
Ankyrin
75
- Short, double helical filaments of F-actin
| - Binds to tail end of spectrin dimers and protein 4.1
Actin
76
- Binds to tail end of spectrin
- Also binds to glycophorin A and C
- Interact with certain membrane phospholipids thus connecting lipid bilayer to cytoskeleton
Protein 4.1
77
- Due to deficiency or abnormalities in structure of spectrin
- Spherical RBCs are more susceptible to osmotic lysis
- Characterized by spherical RBCs, hemolytic anemia and splenomegaly
Hereditary SPHEROCYTOSIS
78
- May be due to abnormalities of spectrin, band 4.1 or glycophorin C
HEREDITARY ELLIPTOCYTOSIS
79
- At least 21 blood group systems
- RBC antigen controlled by genetic locus having a variable number of alleles
- Classification of blood based on the presence or absence of inherited antigenic substances on the surface of red blood cells
Examples: ABO; Rh
BLOOD GROUP SYSTEMS
80
- Are complex oligosaccharides present in most cells of the body and in certain secretions
* RBC – present as glycosphingolipids
* Secretions – present as glycoproteins
- Genes present on long arm of chromosome 9
ABO substance
81
- Found in persons of blood type O
- Precursor of both A and B substance
- Formed by the action of fucosyltransferase that catalyzes the addition of terminal fucose in a 1,2 linkage onto terminal Gal residue of its precursor
H substance
82
A substance vs B substance
A substance - Contains an additional GalNAc
B substance - Contains an additional Gal
83
H gene vs A gene vs B gene
H gene – codes for Fucosyl transferase
A gene – codes for GalNAc transferase
B gene – codes for Gal transferase
84
- Integral membrane protein of RBC
- Gene located at chromosome 1
Clinical Importance:
- Rh(-) pregnant woman who have been previously exposed to Rh(+) blood will develop Abs for Rh factor
- If her infant is Rh(+), Ab can cause lysis of infant’s RBC -> HEMOLYTIC DISEASE OF NEWBORN
Rhesus factor (D antigen)
85
(MNS system)
- Due to polymorphic forms of glycophorin A
- Involves difference in AA sequence at the N terminal of the protein
MN
86
(MNS system)
Subclass of MN group; comprise polymorphic forms of glycophorin B; difference of 1 AA
S
87
HEMOLYTIC ANEMIA
1. Abnormalities outside the RBC membrane
- Hypersplenism
- Transfusion reactions
- Toxins from infectious agents / snake venom
2. Abnormalities within RBC membrane
- Hereditary spherocytosis
- Hereditary elliptocytosis
3. Abnormalities inside the RBC
- Hemoglobinopathy e.g., Sickle cell anemia
- Enzymopathy e.g., G6PD deficiency, Pyruvate kinase deficiency
88
- is used to detect these antibodies or complement proteins that are bound to the surface of red blood cells;
- a blood sample is taken and the RBCs are washed (removing the patient's own plasma) and then incubated with antihuman globulin (also known as "Coombs reagent"). - If this produces agglutination of RBCs, the direct Coombs test is positive, a visual indication that antibodies (and/or complement proteins) are bound to the surface of red blood cells.
direct Coombs test
89
- is used in prenatal testing of pregnant women, and in testing blood prior to a blood transfusion
- detects antibodies against RBCs that are present unbound in the patient's serum. In this case, serum is extracted from the blood, and the serum is incubated with RBCs of known antigenicity.
- If agglutination occurs, the indirect Coombs test is positive
indirect Coombs test
90
Leukocytes
Neutrophil – acute inflammatory response; contains lysosomes for phagocytosis
Lymphocyte –T lymphocyte matures in the thymus mediates cellular immune response – cytotoxic t cells, helper and suppressor t cells;
B lymphocyte – matures in bone marrow mediates humoral immune response, differentiate to plasma cells and produce antibodies
Monocyte – become macrophahges
Eosinophil – defense against parasitic infections (helminthic, protozoan)
Basophil – for allergic reactions and contains heparin, histamine, amines, leukotrines
91
BIOCHEMICAL FEATURES OF NEUTROPHILS
- Active glycolysis
- Active PPP
- Moderate oxidative phosphorylation
- Rich in lysosomes and their degradative enzymes
- Contain unique enzymes (myeloperoxidase, NADPH oxidase) and proteins
- Contain CD11/ CD18 integrins in plasma membrane
92
ACUTE INFLAMMATORY RESPONSE
1. Increased vascular permeability
- By agents release from cells and plasma proteins
- Results in tissue edema
2. Entry of activated neutrophils into tissue
- By chemotactic factors (C5a, peptides from bacteria, leukotrienes)
3. Activation of neutrophils
- Involved turning on of metabolic processes involved in phagocytosis and killing of bacteria
4. Spontaneous subsidence (resolution)
93
Mediated by integrins in neutrophil surface with receptor proteins in endothelial cells
ADHESION OF NEUTROPHILS TO ENDOTHELIAL CELLS
94
- Surface proteins Involved in adhesion to other cells or to specific components of extracellular matrix
- Consist of 2 subunits – α and β – combined at one end to form a ligand-binding extracellular segment (head) and separates into a transmembrane and intracellular domains (stalk)
Integrin
95
- Can bind to more than one ligand and a ligand can be recognized by various integrins (Ligands often contain Arg-Gly-Asp (RGD) sequence)
- Are expressed on a wide variety of cells and most cells express several integrins
Integrins
96
- Deficiency of β5 (C18) subunit of intern
- Characterized by recurrent bacterial and fungal infections
- Adhesion of WBC to endothelial cells is diminished -> lesser number of neutrophils enter tissue to combat infection
LEUKOCYTE ADHESION DEFICIENCY
97
ACTIVATION OF NEUTROPHILS
Similar to platelet activation
Activators
- Via specific receptors
- Interaction with bacteria
- Binding of chemotactic factors
- Ag-Ab complexes
98
- Rapid utilization of oxygen and production of large amount of reactive derivatives e.g O2 •, H2O2, OH •, OCl-, which are potent microbicidal agents
- Involves NADPH:O2-oxidoreductase (NADPH oxidase) and b type cytochrome (Cyt b558 or b245)
Respiratory Burst
99
- Responsible for the green color of pus
H2O2 + X- + H+ → HOX +H2O
Myeloperoxidase
100
- Powerful oxidant, highly microbicidal
- Reacts with primary and secondary amines to produce chloramines which are less powerful oxidants but acts as microbicidal without causing tissue damage
HOCl ( Hypochlorous acid)
101
- Hydrolyzes link between N-acetylmuraminic acid and N-acetylglucosamine found in bacterial cell wall
Lysozyme
102
- Antibiotic peptides of 20-33 amino acids; kills bacteria by causing membrane damage
Defensins
103
- is a globular glycoprotein that is widely represented in various secretory fluids, such as milk, saliva, tears, and nasal secretions
- is also present in secondary granules of PMN and is secreted by some acinar cells. Lactoferrin can be purified from milk or produced recombinantly.
- Human colostrum ("first milk") has the highest concentration
Lactoferrin
104
- Characterized by recurrent infections and widespread granulomas in the skin, lungs and lymph nodes
- Due to a deficiency in NADPH oxidase system
- Granuloma forms as an attempt to wall off bacteria that have not been killed
CHRONIC GRANULOMATOUS DISEASE
105
- Includes elastase, collagenase, gelatinase, cathepsin G, plasminogen activator
- Mostly found in lysosomes
- Can digest proteins in ECM and cause serious tissue damage
- Action kept in check by anti-proteinases present in plasma and ECF (E.g., α1-antiproteinase, α2-macroglobulin)
PROTEINASES IN NEUTROPHILS
106
PROTEINASE-ANTIPROTEINASE BALANCE
- HOCl can activate certain proteinase and inactivate anti-proteinase
- Tissue inhibitors of metalloproteinases and α1-antichymotrypsin can be hydrolyzed by activated elastase
- α1-antiprotease inhibitor can be hydrolyzed by activated collagenase and gelatinase
107
- Consists of water, electrolytes, metabolites, nutrients, proteins, and hormones
- Water and electrolyte composition of plasma is practically the same as that of all ECF
- Complex mix of proteins
Plasma
108
- Plays a major role in the body’s defense mechanisms
- Synthesized by B cells
- Circulating humoral antibodies
- Plasma immunoglobulins are synthesized mainly by plasma cells in response to antigen exposure
IMMUNOGLOBULINS
109
- Total protein in human plasma is approximately 7–7.5 g/dL
- Osmotic or oncotic pressure is exerted by the plasma proteins
- Hydrostatic pressure in the vascular compartment and osmotic/oncotic pressure in the interstitial space drives fluid out of the vessels
- most are synthesized in the liver
- are generally synthesized in membrane-bound polyribosomes
Plasma Protein
110
If PP concentration is decreased, fluid will not flow back into the intravascular compartment which can lead to
EDEMA
111
FUNCTIONS OF PLASMA PROTEINS
- Alpha fetoprotein (AFP)
- Antiprotease
- Blood clotting
- Enzymes
- Hormones
- Immune defense
- Inflammatory responses
- Transport of binding proteins
112
(analysis of plasma protein)
- separate the proteins of the plasma into three major groups—fibrinogen, albumin, and globulins—by the use of varying concentrations of sodium or ammonium sulfate
Salting-out method
113
(analysis of plasma protein)
- Most common method of analyzing plasma proteins
- Cellulose acetate is widely used as a supporting medium
Electrophoresis
114
Plasma Proteins
- Most plasma proteins except for albumin are glycoproteins
- Many plasma proteins exhibit polymorphism- genetic variation seen in at least 2 phenotypes
- The ABO blood group substances are the best-known examples of human polymorphisms
115
- Plasma proteins that increase during acute inflammatory states or secondary to tissue damage
e. g. C-reactive protein (CRP, so-named because it reacts with the C polysaccharide of pneumococci), α1-antitrypsin, haptoglobin, α1-acid glycoprotein, and fibrinogen
Acute Phase Proteins
116
- is used as a marker of tissue injury, infection, and inflammation
- Possible predictor of certain cardiovascular conditions secondary to atherosclerosis
C Reactive Protein
117
- Major protein in human plasma
- Makes up about 60% of plasma protein
- 40% of __ is in plasma; the rest is in the extracellular space
- Liver produces 12 g __ per day
Albumin
118
ALBUMIN STRUCTURE
- Mature human albumin has 1 polypeptide chain of 585 amino acids and contains 17 disulfide bonds
- Divided into 3 domains
- Ellipsoidal in shape; does not increase the viscosity of plasma as much as elongated proteins do
- Responsible for 75-80% of plasma osmotic pressure
119
ALBUMIN SYNTHESIS
- Initially formed as a preproprotein
- Decreased synthesis in liver disease
- Patients with liver disease show a decrease in plasma albumin to globulin ratio
- Decreased in Kwashiorkor (protein malnutrition)
120
ALBUMIN FUNCTIONS
- Albumin binds with FFA, Ca, Steroids, Bilirubin
- Plays a role in copper transport
- Drugs like sulfonamides, Pen G, and Aspirin are bound to albumin and affect their pharmacologic activity
121
- Plasma glycoprotein that binds extracorpuscular hemoglobin (Hb) in a tight noncovalent complex (Hb-Hp)
* 10% of Hb degraded each day is extra corpuscular
- Free Hb passes through the glomerulus, enters the tubules, and tends to precipitate, Hp prevents loss of free hemoglobin and conserves iron stores
HAPTOGLOBIN (Hp)
122
STRUCTURE OF Hp
- Has 3 polymorphic forms
| - Polymorphism of Hp may be associated with various inflammatory diseases
123
HAPTOGLOBIN (Hp)
When Hp is bound to Hb, it is cleared faster from the circulation
- Half-life of Hb-Hp is 90 minutes compared to 5 days for
Hp alone
- Hb-Hp is catabolized by the liver and iron is conserved and reused
- Low Hp levels are associated with Hemolytic anemia
124
- plays a role in transporting/ transferring iron around the body to sites where it is needed
- Synthesized in the liver
- Concentration of transferrin in plasma is approximately 300 mg/dL
- It can bind 300 μg of iron per deciliter, - represents the Total iron-binding capacity of plasma
Transferrin (Tf)
125
- Normally is only 1/3 saturated with iron
- In iron deficiency anemia, the protein is even less saturated with iron
- In excess storage of iron in the body (eg,Hemochromatosis) the saturation with iron is much greater than one-third
TRANSFERRIN
126
- Contains approximately 23% iron, and apoferritin (the protein moiety free of iron)
- In hemochromatosis, body stores of iron are greatly increased and much more ferritin is present in the tissues, such as the liver and spleen
FERRITIN
127
- Autosomal Recessive disorder
- Caused by MUTATIONS IN THE HFE GENE, which regulates the amount of iron absorbed from food
- A person who inherits a defective gene from each parent will develop hemochromatosis
- A person who inherits a defective gene from one parent is a carrier but usually does not develop the disease. Carriers, however, might have a slight increase in iron absorption.
HEREDITARY HEMOCHROMATOSIS
128
- Appears to be a partly degraded form of ferritin but still containing iron
- Product of RBC hemolysis
- Presence is determined histologically when excessive storage of iron occurs
HEMOSIDERIN
129
- is an α2-globulin
- Has a blue color because of its high copper content
- Carries 90% of the copper present in plasma
- Synthesized in the liver
Ceruloplasmin
130
Ceruloplasmin vs Albumin
Ceruloplasmin binds six atoms of copper very tightly, so that the copper is not readily exchangeable
Albumin carries the other 10% of the plasma copper but binds the metal less tightly than does ceruloplasmin; more important than ceruloplasmin in copper transport because it binds copper less avidly
131
Low levels of ceruloplasmin are found in __ (hepatolenticular degeneration), a disease due to abnormal metabolism of copper
Wilson disease
132
Copper
Excess copper cause problems because it oxidizes proteins and lipids, bind to nucleic acids, and enhance the production of free radicals
The body of the normal adult contains about 100 mg of copper, located mostly in bone, liver, kidney, and muscle
133
- X linked recessive
- expression in all tissues except liver
- onset at Birth
- Sxs: epicathus, thin breakable metallic hair strands. Abnormal torsion of hair to its own axis ( pili torti) vs normal strand
Menkes Disease
134
- Genetic disease in which copper fails to be excreted in the bile and accumulates in liver, brain, kidney and RBC
- Kayser-Fleischer ring - green or golden pigment ring around the cornea due to deposition of copper in Descemet’s membrane
Wilson's Disease
135
- Major component of the α1 fraction of human plasma
- Synthesized by hepatocytes and macrophages
- Principal serine protease inhibitor of human plasma
- The major genotype is MM, and its phenotypic product is PiM
- Deficiency is associated with EMPHYSEMA AND ALPHA 1 ANTITRYPSIN DEFICIENCY DISEASE
α-1-Antiproteinase (α-1-Antitrypsin)
136
- When α1-antitrypsin is deficient and PMN WBC increase in the lung (such as during pneumonia), the affected individual lacks a countercheck to proteolytic damage of the lung by proteases such as elastase
- Methionine of α1-antitrypsin is involved in its binding to proteases
- Smoking oxidizes this methionine to methionine sulfoxide and thus inactivates
Emphysema
137
- A major member of a group of plasma proteins that include complement proteins C3 and C4
- These proteins contain a unique internal cyclic thiol ester bond (formed between a cysteine and a glutamine residue) and for this reason have been designated as the Thiol ester plasma protein family
- binds many proteinases and is thus an important panproteinase inhibitor
- Approximately 10% of the zinc in plasma is transported by α2-macroglobulin, the remainder being transported by albumin
α2-Macroglobulin
138
- Accumulation of insoluble fibrillar proteins
- Amyloid disorders include Alzheimer's disease and other neurodegenerative diseases, and transmissible prion diseases (Kuru)
Amyloidosis
139
- Contain a min. of 2 identical light (L) chains and 2 identical heavy (H) chains, held together as a tetramer (L2H2) by disulfide bonds
- The half of the light (L) chain toward the carboxyl terminal is referred to as the constant region (CL), while the amino terminal half is the variable region of the light chain (VL)
IMMUNOGLOBULINS
140
STRUCTURE OF IMMUNOGLOBULIN G (IgG)
- Each light chain consists of a variable (VL) and a constant (CL) region
- Hinge region confers flexibility in binding to antigenic sites
- Digestion of an immunoglobulin by the enzyme papain produces two antigen-binding fragments (Fab) and one crystallizable fragment (Fc), which is responsible for functions of Ig
- Each heavy chain consists of a variable region (VH) and a constant region (CH) that is divided into three domains (CH1, CH2, and CH3)
141
CH2 vs CH3
The CH2 domain contains the complement-binding site
CH3 domain contains a site that attaches to receptors on neutrophils and macrophages
142
The antigen-binding site is formed by the hypervariable regions, also called __
Complementarity-Determining Regions (CDRs)
143
- Responsible for the class-specific effector functions of the different immunoglobulin molecules
- IgG exist only in the basic tetrameric structure
- IgA and IgM exist as higher order polymers of two, three (IgA), or five (IgM) tetrameric units
CONSTANT REGIONS
144
Both IgA and IgM have a J chain, but only secretory __ has a secretory component
IgA
145
- Type of H chain determines the class of Ig and its effector function
- 5 classes of H chain: γ, α, μ, δ, and ε
- μ and ε chains each have 4 CH domains rather than 3
5 immunoglobulin classes: IgG, IgA, IgM, IgD, and IgE
THE HEAVY (H) CHAIN
146
Light and heavy chains
- The L chains and H chains are synthesized as separate molecules and assembled within the B cell or plasma cell into mature immunoglobulin molecules
- Both light and heavy chains are products of multiple genes
147
- diversity depends on gene rearrangements
| - Each person is capable of generating __ directed against perhaps 1 million different antigens
Antibody
148
Immunoglobulins
1. IgG - Main antibody in the secondary response. Opsonizes bacteria, making them easier to phagocytose. Fixes complement, which enhances bacterial killing. Crosses the placenta.
2. IgA - secretory IgA prevents attachment of bacteria and viruses to mucous membranes. Does not fix complement.
3. IgM - Produced in the primary response to an antigen. Fixes complement. Does not cross the placenta. Antigen receptor on the surface of B cells.
4. IgD - Found on the surfaces of B cells where it acts as a receptor for antigen.
5. IgE - Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to antigen (allergen). Defends against worm infections by causing release of enzymes from eosinophils.
149
- Overproduction and underproduction of Immunoglobulins may result in disease states
- Severe reduction in synthesis of an immunoglobulin class due to a genetic abnormality can result in a serious immunodeficiency disease
Immunodeficiency
150
- Antibodies that are identical
- Produced by one type of immune cell that are all clones of a single parent cell
- Made by fusing the spleen cells from a mouse or rabbit that has been immunized with the desired antigen with myeloma cells
Monoclonal antibodies (mAb or moAb)
151
- Component of the innate immune system
- Series of plasma enzymes, regulatory proteins, and proteins that are activated in a cascading fashion, resulting in cell lysis
- The complement system comprises about 20 Plasma Proteins
The Complement System
152
Pathways of the Complement System
Classic activation pathway - activated by antigen/antibody immune complexes
MBL activation pathway - activated by microbes with terminal mannose groups
Alternative activation pathway - activated by microbes or tumor cells
Terminal pathway - common to the pathways and leads to the membrane attack complex that lyses cells
153
Major components of the Immune system:
(1) Humoral immunity (B cells)
(2) Cell-mediated immunity (T cells, monocytes)
(3) Phagocytic cells of the reticuloendothelial system (macrophages), as well as polymorphonuclear leukocytes
(4) Complement
154
Defects in the Immune system
DEFECTS IN CELLULAR IMMUNITY generally result in viral, mycobacterial, and fungal infections
- An extreme deficiency in cellular immunity is AIDS
ANTIBODY DEFICIENCIES result in recurrent bacterial infections, frequently with organisms such as S. pneumoniae and Haemophilus influenzae
155
Defects in the Immune system 2
DISORDERS OF PHAGOCYTE FUNCTION are frequently manifested by recurrent skin infections, often due to Staphylococcus aureus
DEFICIENCIES OF EARLY AND LATE COMPLEMENT COMPONENTS ARE associated with autoimmune phenomena and recurrent Neisseria infections
156
Glycobiology vs Glycome vs Glycomics
Glycobiology - the study of the roles of sugars in health and disease
Glycome - the entire complement of sugars, whether free or present in more complex molecules, of an organism
Glycomics - an analogous term to genomics and proteomics, is the comprehensive study of glycomes, including genetic, physiologic, pathologic, and other aspects
157
Glycosylation vs Glycation
Glycosylation - enzymic attachment of sugars; most frequent post-translational modification of proteins
Glycation - Nonenzymic attachment of sugars to proteins
158
- proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide backbones two to ten sugar residues in length
- glycoproteins do not have serial repeats
- Carbohydrate chains are often branched instead of linear
contain highly variable amounts of carbohydrate
Glycoproteins
159
Glycoproteins vs Proteoglycans
Glycoproteins - Proteins conjugated to saccharides lacking a serial repeat unit
Protein >> carbohydrate
Proteoglycans - Proteins conjugated to polysaccharides with serial repeat units
- Glycosaminoglycans - Repeat unit
- Mucopolysaccharides - HexN and HexUA
- Carbohydrate >> protein
160
- participate in a broad range of cellular phenomena
* cell surface recognition
* cell surface anti genicity
* components of the extracellular matrix
* mucins of the gastrointestinal and urogenital tracts
* Constitute almost all of the globular proteins present in human plasma
Glycoproteins
161
Some Functions of the Oligosaccharide Chains of Glycoproteins
- Modulate physicochemical properties, eg, solubility, viscosity, charge, conformation, denaturation, and binding sites for various molecules, bacteria viruses and some parasites
- Protect against proteolysis, from inside and outside of cell
- Affect proteolytic processing of precursor proteins to smaller products
- Are involved in biologic activity, eg, of human chorionic gonadotropin (hCG)
- Affect insertion into membranes, intracellular migration, sorting and secretion
- Affect embryonic development and differentiation
- May affect sites of metastases selected by cancer cells
162
Principal Sugars Found in Glycoprotein
About 200 monosaccharides are found in nature; however, only eight are commonly found in the oligosaccharide chains of glycoproteins.
N- ACETYLNEURAMINIC ACID (NeuAc) is usually found at the termini of oligosaccharide chains, attached to subterminal galactose (Gal) or N -ACETYLGALACTOSAMINE (GalNAc) residues. The other sugars listed are generally found in more internal positions.
Sulfate is often found in glycoproteins, usually attached to Gal, GalNAc, or GlcNAc.
163
Nucleotide Sugars Act as Sugar Donors in Many Biosynthetic Reactions
- in most biosynthetic reactions, it is the nucleotide sugar that is involved in such reactions
* uridine diphosphate glucose (UDP-Glc)
- Most nucleotide sugars are formed in the cytosol
164
- An enzyme that catalyzes the hydrolysis of a bond joining a sugar of a glycoside to an alcohol or another sugar unit
useful in examining structural and functional aspects of glycoproteins
- act at either external (exoglycosidases) or internal (endoglycosidases) positions of oligosaccharide chains
glycosidases
165
- Isolated in liver cells
- recognizes the Galactose moiety of many desialylated plasma proteins
- leads to their endocytosis
Mammalian Asialoglycoprotein Receptor
166
- are carbohydrate-binding proteins that agglutinate
cells or precipitate glycoconjugates; a number of lectins are
themselves glycoproteins
- contain at least two sugar-binding sites; proteins with only a single sugar-binding site will not agglutinate cells or precipitate glycoconjugates
Lectins
167
Plant lectins were formerly called __, because of their ability to agglutinate red blood cells by reacting with the cell surface glycoproteins.
phytohaemagglutinins
168
STRUCTURE OF GLYCOPROTEIN OLIGOSACCHARIDES
oligosaccharide components of glycoproteins
- branched heteropolymers composed primarily of D-hexoses
- neuraminic acid
- L-fucose
169
Structure of the linkage between carbohydrate and protein
- attached to the protein through an N-glycosidic link (the sugar chain is attached to the AMIDE GROUP OF an ASPARAGINE SIDE CHAIN)
- attached to the protein through an O-glycosidic link
(the sugar chain is attached to to the HYDROXYL GROUP OF either a SERINE OR THREONINE R-GROUP)
170
Three Major Classes of Glycoproteins
1. O-glycosidic linkage - involving the hydroxyl side chain of serine or threonine and a sugar such as N-acetylgalactosamine (GalNAc-Ser[Thr]
2. N-glycosidic linkage - Involving amide nitrogen of asparagine and N -acetylglucosamine (GlcNAc-Asn)
3. glycosylphosphatidylinositol-anchored (GPI-anchored, or GPI-linked) glycoproteins
- linked to the carboxyl terminal amino acid of a protein via a phosphoryl-ethanolamine moiety joined to an oligosaccharide (glycan), which in turn is linked via glucosamine to phosphatidylinositol (PI)
171
- may have one or more of a wide variety of sugars arranged in either a linear or a branched pattern
- found in extracellular glycoproteins or as membrane glycoprotein components (O-linked oligosaccharides on the surface of RBCs)
- Tend to be built one residue at a time in Golgi
- More extensive netowrk than N-linked
O-Linked oligosaccharides
172
- Chain of 14 residues as a polypeptidecomes off ribosome in RER
- At Golgi apparatus, glycosidases remove some residues; glucosyltransferases add new monosaccharides
- Highly specific process
- two classes: Complex and High-mannose
- core pentasaccharide
- High-mannose - mannose (Man)
- complex oligosaccharides contain a diverse group of additional sugars
N-acetyl-glucosamine (GlcNAc)
L-fucose (Fuc),
N-acetylneuraminic acid (NANA)
- Gives tremendous variety and specificity to molecules
N-linked oligosaccharides
173
SYNTHESIS OF GLYCOPROTEINS
- synthesized on ribosomes attached to the RER
- Molecular “address labels”
- Proteins directed to the RER
- Extruded to the lumen
- transported via secretory vesicles to the Golgi complex
- integrated into the Golgi membrane
174
Carbohydrate components of Glycoproteins
```
1. nucleotide sugars
UDP-glucose
UDP-galactose
UDP N-acetylglucosamine
UDP N-acetylgalactosamine
```
2. donate sugars
GDP-mannose
GDP-L-fucose
CMP-N-acetylneuraminic acid
3. Covalently attached to specific amino acid R-groups of the protein
175
Synthesis of O-linked glycosides
- synthesized on the RER
- Extruded to the lumen
- transfer of an N-acetylgalactosamine (from UDP-N-acetylgalactos amine) onto the R-group of a specific serine or threonine
- Role of glycosyltransferases
176
Role of glycosyltransferases
1. Responsible for the stepwise synthesis of the oligosaccharides
2. bound to the membranes of the Golgi apparatus
3. recognize the actual structure of the growing oligosaccharide as the appropriate substrate
177
Synthesis of the N-linked glycosides
- occurs in the lumen of the ER
- requires the participation of dolichol pyrophosphate
* the phosphorylated form of dolichol
* lipid of the ER membrane 80-100 carbons long
- Processed in the ER and Golgi
178
- synthesized on the RER and enters its lumen
- does NOT become glycosylated with individual sugars
- a lipid-linked oligosaccharide is first constructed
consists of dolichol attached through a pyrophosphate linkage to an oligosaccharide
- sugars added sequentially to the dolichol by the membrane-bound glycosyltransferases (N-acetylglucosamine; Mannose; glucose)
1. Synthesis of dolichol-linked oligosaccharide
179
- processed by the removal of specific mannosyl and glucosyl residues as the glycoprotein moves through the RER
- Oligosaccharide chains are completed in the Golgi by addition of a variety of sugars to produce a complex glycoprotein (N-acetylglucosamine; N-acetylgalactosamine
additional mannoses; fucose or NANA as terminal groups)
- OR they are not processed further
branched, mannose-containing chains in a high-mannose glycoprotein
2. Final processing of N-linked oligosaccharides
180
Fates of N-linked and O-linked Glycoproteins
- Same as O-linked (released by the cell; become part of a cell membrane)
- N-linked glycoproteins can be translocated to the lysosomes
181
LYSOSOMAL DEGRADATION OF GLYCOPROTEINS
- lysosomal acid hydrolases generally specific for the removal of one component of the glycoprotein
- Exoenzymes
- “last on, first off”
182
- very rare, autosomal recessive genetic diseases
- caused by a deficiency of any one of the degradative enzymes
- Results in accumulation of partially degraded structures in the lysosomes
- α-mannosidosis type 1 (Deficiency of enzyme α-mannosidase)
glycoprotein storage diseases (oligosaccharidoses)
183
- linked glycoproteins comprise the third major class of glycoprotein.
- structure (sometimes called a "sticky foot") involved in linkage of the enzyme acetylcholinesterase (ACh esterase) to the plasma membrane of the red blood cell
Glycosylphosphatidylinositol (GPI)
184
GPI-linked proteins are anchored to the outer leaflet of the plasma membrane by the fatty acids of __
phosphatidylinositol (PI)
185
Three possible functions of GPI
1. greatly enhanced mobility of a protein in the plasma membrane
2. may connect with signal transduction pathways
3. can target certain proteins to apical domains and also basolateral domains of the plasma membrane of certain polarized epithelial cells
186
- assembled independently by a series of enzyme-catalyzed reactions
- transferred to the carboxyl terminal end of its acceptor protein
- cleavage of the preexisting carboxyl terminal hydrophobic peptide
Glypiation
187
- Thought to Be Important in the Causation of Tissue Damage in Diabetes Mellitus
- End-products of glycation (non-enzymic attachment of sugars (mainly glucose) to amino groups of proteins)
- Maillard reaction
Advanced Glycation End-Products (AGEs)
188
- Schiff bases
- intermediate products
- Amadorirearrangement to ketoamines
- involved in the browning of certain foodstuffs that occurs on storage or processing (eg, heating)
Maillard reaction
189
Glycoproteins Are Involved in Many Biologic Processes and in Many Diseases
- Fertilization glycoprotein
ZP3 is an O-linked glycoprotein that functions as a sperm
receptor
- Inflammation and Lymphocyte Homing
190
- is a rare condition probably due to mutations affecting the activity of a Golgi-located GDP-fucose transporter.
- It can be considered a congenital disorder of glycosylation.
- Subjects suffer life-threatening, recurrent bacterial infections and also psychomotor and mental retardation.
- The condition appears to respond to oral fucose.
Leukocyte adhesion deficiency (LAD) II
191
—congenital dyserythropoietic anemia type II—is another disorder in which abnormalities in the processing of N-glycans are thought to be involved. Some cases have been claimed to be due to defects in alpha–mannosidase II
Hereditary erythroblastic multinuclearity with a positive acidified lysis test (HEMPAS)
192
is an acquired mild anemia characterized by the presence of hemoglobin in urine due to hemolysis of red cells, particularly during sleep. This latter phenomenon may reflect a slight drop in plasma pH during sleep, which increases susceptibility to lysis by the complement system
Paroxysmal nocturnal hemoglobinuria (PNH)
193
- serves to target enzymes into the lysosome.
Mannose 6-phosphate
194
- is an uncommon condition characterized by severe progressive psychomotor retardation and a variety of physical signs, with death often occurring in the first decade of life.
- Cells lack almost all of the normal lysosomal enzymes; the lysosomes thus accumulate many different types of undegraded molecules, forming inclusion bodies.
I-cell disease
195
- binds to cell surface glycoprotein receptor molecules containing N-acetylneuraminic acid via a hemagglutinin protein. The virus also has a neuraminidase that plays a key role in allowing elution of newly synthesized progeny from infected cells. If this process is inhibited, spread of the viruses is markedly diminished.
Influenza virus A
196
- the cause of AIDS, attaches to cells via one of its surface glycoproteins (gp 120) and uses another surface glycoprotein (gp 41) to fuse with the host cell membrane.
Human immunodeficiency virus type 1 (HIV-1)
197
- is the major cause of peptic ulcers. It binds to at least two different glycans present on the surfaces of epithelial cells in the stomach allowing it to establish a stable attachment site to the stomach lining
Helicobacter pylori
198
- complex ECM where most mammalian cells are located
- protects the organs
- provides elasticity (blood vessels, lungs, and skin)
Connective tissue
199
Connective Tissue contains three major classes of biomolecules
1. collagen, elastin, and fibrillin, certain specialized proteins
2. fibronectin and laminin embedded in
3. proteoglycan
200
- Most abundant protein in the world
- major component of most connective tissues (Organizes and strengthens extracellular matrix)
- Extensively modified by posttranslational
modification
- constitutes approximately 25% of the protein of mammals
- At least 28 distinct types
- made up of over 30 distinct polypeptide chains
Collagen
201
* Most common (90%)
* Seen in: Bone (made by osteoblasts); Skin; Tendon; Dentin; fascia; Cornea; late wound repair
* Clinical Correlation: ↓ production in OSTEOGENESIS IMPERFECTA TYPE I
*bONE
Type I Collagen
202
```
• Seen in: Cartilage (including hyaline); vitreous body (eye);
nucleus pulposus (lumbar disc)
```
*carTWOlage
Type II Collagen
203
* Seen in: Reticulin—skin, blood vessels; Uterus; Fetal tissue; granulation tissue
* Clinical Correlation: VASCULAR TYPE OF EHLERS-DANLOS SYNDROME
*threE D - Ehlers Danlos
Type III Collagen
204
• Seen in:Basement membrane; basal lamina; Lens (eye)
• Clinical Correlates: Defective in Alport syndrome; targeted by autoantibodies in Goodpasture syndrome (rare disease that can involve quickly worsening kidney failure
and lung disease)
*“Type Four is under the floor”
Type IV Collagen
205
- have a triple helical structure
- occurrence of glycine residues at every third position of the triple helical portion of the alpha chain
* the only amino acid small enough to be accommodated in the limited space available in the central core of the triple helix
* Gly-X-Y
Collagen
206
- confer rigidity on the collagen molecule
- Prolyl hydroxyls
- ascorbic acid (vitamin C) and α-ketoglutarate
- lysol hydroxylase (further modified by the addition of galactose or galactosyl-glucose through an O-glycosidic linkage)
Proline and hydroxyproline
207
Synthesis of Collagen: Inside fibroblasts (1-4)
1. SYNTHESIS (RER): Translation ofcollagen alpha chains (procollagen) - usually Gly-X-Y (X and Y are proline or
lysine) . Glycine content best reflect collagen synthesis (Collagen is ⅓ glycine).
2. HYDROXYLATION (RER): Hydroxylation Of specific proline and lysine residues (requires Vitamin C; deficiency:SCURVY)
3. GLYCOSYLATION (RER): Glycosylation of pro-alpha-chain hydroxylysine residues and formation of pro collagen via hydrogen and disulfide bonds (triple helix of 3 collagen alpha chains). Problems forming triple helix: OSTEOGENESIS IMPERFECTA
4. EXOCYTOSIS: Exocytosis of pro collagen into extracellular space
208
Synthesis of Collagen: Outside fibroblasts (5-6)
5. PROTEOLYTIC PROCESSING: Cleavage of disulfide-rich terminal regions of procollagen, transforming it into
insoluble tropocollagen
6. CROSS-LINKING: Reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysine cross linkage (by copper-containing lysol oxidase) to make collagen fibrils. Problems with cross-linking: EHLERS-
DANLOS SYNDROME, MENKES DISEASE
209
- Aka Cutis hyperelastica
- group of inherited disorders
- clinical features: hyper hyperextensibility of the skin; abnormal tissue fragility; increased joint mobility
Types
o Classical type - joint and skin symptoms; caused by a
mutation in type V collagen
o Vascular type - most serious; vascular and organ rupture;
deficient type III collagen
Ehlers-Danlos syndrome
210
- type IV collagen
- Clinical features: Hematuria; ocular lesions; hearing loss
- patients may eventually develop endstage renal disease
- Electron microscopy: characteristic abnormalities of the structure of the basement membrane and lamina densa
Alport syndrome (hereditary nephritis)
211
* the skin breaks and blisters as a result of minor trauma
* The dystrophic form is due to mutations in COL7A1, affecting the structure of type VII collagen (forms delicate fibrils that anchor the basal lamina to collagen fibrils in the dermis)
Epidermolysis bullosa
212
- another variant, is due to mutations in keratin 5
Epidermolysis bullosa simplex
213
• affects the structure of collagen
• due to a deficiency of ascorbic acid (vitamin C)
• Major signs are bleeding gums, subcutaneous hemorrhages, and poor wound healing.
• due to reduced activity of the enzymes prolyl and lysyl
hydroxylases, both of which require ascorbic acid as a cofactor and are involved in posttranslational
modifications which give collagen molecules rigidity.
Scurvy
214
- X-linked recessive
- impaired copper absorption and transport
- due to defective Menkes protein (ATP7A)
- results in defective cross-linking of collagen and elastin (copper dependent enzyme lysyl oxidase)
- Clinical Features: brittle, “kinky” hair; growth retardation; hypotonia
Menkes disease
215
- responsible for properties of extensibility and elastic recoil in tissues (lung, large arterial blood vessels, and some elastic ligaments)
- appears to be only one genetic type
- synthesized as a soluble monomer of ∼70 kDa called TROPOELASTIN
- hydroxylated to hydroxyproline by prolyl hydroxylase
Elastin
216
- major cross-links formed in elastin
| - Results in insolubility and stability once cross-linked
Desmosines
217
- Seen in 90% of pxs with deletions in the elastin gene
located at 7q11.23
- developmental disorder affecting connective tissue and the central nervous system
- supravalvular aortic stenosis
Williams-Beuren syndrome
218
Decreased elastin also seen in:
- pulmonary emphysema
- cutis laxa
- aging of the skin
219
- large glycoproteins (about 350 kDa)
- secreted into the ECM by fibroblast
- Fibrillin-1 - main fibrillin present
- Fibrillin-2 - thought to be important in deposition of microfibrils early in development
Fibrillin
220
Structural components of microfibrils
- fine fiber-like strands 10 to 12 nm in diameter
- provide a scaffold for the deposition of elastin in the ECM
- found in elastic fibers and also in elastin-free bundles in the eye, kidney, and tendons
221
- Autosomal dominant
- Most cases caused by mutations in the gene (on chromosome 15) for fibrillin-1
- Affects numerous organs:
* eyes - causing dislocation of the lens, known as ectopia lentis)
* skeletal system - tall patients exhibiting long digits [arachnodactyly] and hyperextensibility of the joints
* cardiovascular system - weakness of the aortic media, leading to dilation of the ascending aorta
Marfan syndrome
222
- found in a soluble form in plasma
- two identical subunits joined by two disulfide bridges near their carboxyl terminals
- contains an Arg-Gly-Asp (RGD) sequence
- Contains three types of repeating motifs (I, II, and III)
FIBRONECTIN
223
- Major protein component of basal laminas (specialized areas of the ECM that surround epithelial and some other cells (eg, muscle cells))
- Consists of three distinct elongated polypeptide chains (α, β, and γ chains) linked together to form a complex, elongated shape
- forms networks in basal laminas which are attached to type IV collagen networks
LAMININ
224
- basal lamina layers make up the glomerular membrane
1. important Pores not allowing molecules >8nm to pass
2. Negative charged surface of heparan sulfate repel simlarly charged substances
renal glomerulus
225
– caused by antibodies directed against various components of the glomerular membrane
– This alters the pores and the amounts and dispositions of the negatively charged macromolecules referred to above,and relatively massive amounts of albumin (and of certain other plasma proteins) can pass through into the urine, resulting in severe albuminuria.
Glomerulonephritis
226
- large complexes of negatively charged heteropolysaccharide chains
- generally associated with a small amount of protein (“core protein”), forming proteoglycans >> typically consist of up to 95% carbohydrate
Glycosaminoglycans (GAGs)
227
- have the special ability to bind large amounts of water
- Produces the gel-like matrix that forms the basis of the body’s ground substance
- make up the extracellular matrix (ECM) along with collagen, elastin, and fibrillin-1, and adhesive proteins such as fibronectin
Glycosaminoglycans (GAGs)
228
STRUCTURE OF GLYCOSAMINOGLYCANS
- long, unbranched, heteropolysaccharide chains composed of a repeating disaccharide unit [acidic sugar–amino sugar]n
* amino sugar is either D-glucosamine or D-galactosamine
* acidic sugar is either D-glucuronic acid or its C-5 epimer L-iduronic acid
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Types of GAGs
- hyaluronic acid (hyaluronan)
- chondroitin sulfate
- keratan sulfates I and II
- heparin
- heparan sulfate
- dermatan sulfate
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Structure of proteoglycan monomers
- Found in every tissue of the body, mainly in the ECM or ground substance
- consists of a core protein to which up to 100 linear chains of GAGs are covalently attached (Chains may each be composed of up to 200 disaccharide units)
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Biosynthesis of Glycosaminoglycans
1. Attachment to Core Proteins
2. Chain Elongation
3. Chain Termination
4. Further Modifications
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- occurs in the endoplasmic reticulum
3 types:
1. O-glycosidic bond between xylose (Xyl) and Ser
2. O-glycosidic bond forms between GalNAc (N-acetylgalactosamine) and Ser (Thr)
3. N-glycosylamine bond between GlcNAc (N-acetylglucosamine) and the amide nitrogen of Asn
Attachment to Core Proteins
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- “one enzyme, one linkage”
- transfer of a xylose from UDP-xylose to the hydroxyl group of a serine (or threonine)
* catalyzed by xylosyl transferase
* most common linkage region
- Addition of Two galactose molecules
- Sequential addition of alternating acidic and amino sugars
Chain Elongation
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result from:
1. sulfation, particularly at certain positions of the sugars, and
2. the progression of the growing GAG chain away from the membrane site where catalysis occurs
Chain Termination
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Further Modifications
Sulfotransferases
- Golgi-located enzymes
- Introduces sulfate groups onto GalNAc and other moieties
- use 3′-phosphoadenosine-5′- phosphosulfate [PAPS; active sulfate] as the sulfate donor
Wpimerase
- Catalyzes epimerization of GlcUA to IdUA residues
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Degradation of GAGs
Lysosomal hydrolyses - endoglycosidases, exoglycosidases, and sulfatases
slow turnover - T1/2 days to weeks
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- Result from deficiencies of enzymes that degrade Glycosaminoglycans
- Chronic; Progressive; Multiorgan disease
Common Clinical Features:
- organomegaly (eg, hepato- and splenomegaly;
- severe abnormalities in the development of cartilage and bone;
- abnormal facial appearance
- mental retardation
- defects in hearing, vision
- cardiovascular system abnormalities
Mucopolysaccharidoses (MPSs)
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Enzyme affected: α-L-Iduronidase
GAG Affected: Dermatan sulfate, heparan sulfate
S/sx: Mental retardation, coarse facial features, hepatosplenomegaly, cloudy cornea
Hurler-, Scheie-Hurler-Scheie syndrome
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enzyme affected: Iduronate sulfates
GAG affected: Dermatan sulfate, heparan sulfate
s/sx: Mental retardation
Hunter syndrome
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- Mineralized connective tissue
- Contains 99% of body’s calcium
- contains both organic and inorganic material:
1. Inorganic - Crystalline hydroxyapatite—Ca10(PO4)6(OH)
- Confers strength and resilience
- sodium; Magnesium; Carbonate; fluoride
2. Organic - type I collagen (90-95%)
Bone
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- found in mature bone
- involved in the maintenance of the bone matrix
- descended from osteoblasts
- T1/2= 25 years
Osteocytes
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- multinucleated cells derived from pluripotent hematopoietic stem cells
- plays a key role in bone resorption
Osteoclasts
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- mononuclear cells derived from pluripotent mesenchymal precursors
- responsible for the deposition of the new bone matrix (osteoid) and its subsequent mineralization
- control mineralization by regulating the passage of calcium and phosphate ions across their surface membranes
Osteoblasts
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- an enzyme in bone cell membranes
- generates phosphate ions from organic phosphates
- contributes to mineralization
Alkaline phosphatase
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- “brittle bone disease”
- Eight types (I-VIII)
- Genetic bone disorder caused by a variety of gene defects
- autosomal dominant (Most common)
Clinical Features:
- Multiple fractures with minimal trauma
- may occur during the birth process
- Blue sclerae due to the translucency of the connective tissue over the choroidal veins
- Hearing loss (abnormal ossicles)
- Dental imperfections due to lack of dentin
Osteogenesis Imperfecta
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- “marble bone disease”
- characterized by increased bone density
- Caused by inability to resorb bone due to mutations in the gene (located on chromosome 8q22)
- Encodes carbonic anhydrase II (CA II)
defect in bone turnover characteristically results in skeletal fragility despite increased bone mass
Osteopetrosis
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- generalized progressive reduction in bone tissue mass per unit volume
- Results in skeletal weakness
2 Types:
1. primary type 1 - commonly occurs in women after menopause
2. primary type 2 - senile; occurs in both sexes post 75 years
more prevalent in women (ratio 2:1 female:male)
Osteoporosis
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Stages in Hemostasis
1. Vascular constriction
2. Formation of a temporary, loose platelet plug
3. Clot formation
4. Dissolution of the clot
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The process of blood clotting (thrombus formation) and then the subsequent dissolution of the clot, following repair of the injured tissue
Hemostasis
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Types of thrombi or clots
1. white thrombus
- is composed of platelets and fibrin and is relatively poor in erythrocytes
- It forms at the site of an injury or abnormal vessel wall, particularly in areas where blood flow is rapid (arteries)
2. The red thrombus
- consists of red cells and fibrin
- may form in vivo in areas of retarded blood flow or stasis (eg, veins)
- may form at a site of injury or in an abnormal vessel in conjunction with an initiating platelet plug
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is initiated within 20 seconds after an injury occurs to the blood vessel damaging the endothelial cells
Coagulation
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Coagulation
Primary hemostasis - Platelets form a hemostatic plug at the site of injury
Secondary homeostasis - Occurs when coagulation factors respond (in a complex cascade) to form fibrin strands which strengthen the platelet plug
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Platelet Activation and von Willebrand Factor (vWF)
- In order for hemostasis to occur, platelets must adhere to exposed collagen, release the contents of their granules, and aggregate
- The adhesion of platelets to the collagen exposed on endothelial cell surfaces is mediated by von Willebrand factor (vWF)
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The function of __ is to act as a bridge between a specific glycoprotein on the surface of platelets and collagen fibrils
von Willebrand factor (vWF)
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Platelet
Bernard-Soulier disease – defect of platelet adhesion
Glanzmann’s thrombastenia – defect of platelet aggregation
Thrombocytopenia =
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The Coagulation Cascade
1. Contact Activation pathway - formerly known as the Intrinsic Pathway
2. Tissue Factor pathway - formerly known as the Extrinsic pathway - primary pathway for the initiation of blood coagulation
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- Series of reactions, in which a zymogen (inactive enzyme precursor) of a serine protease and its glycoprotein co-factor are activated ultimately resulting in cross-linked fibrin
- Coagulation factors are generally indicated by Roman numerals, with a lowercase (a) appended to indicate an active form
The Coagulation Cascade
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- Exposure of collagen to a vessel surface is the stimulus for the contact phase
- Results in conversion of prekallikrein to kallikrein, which in turn activates factor XII to factor XIIa
- Factor XIIa hydrolyze more prekallikrein to kallikrein and activates factor XI to factor XIa and leads to the release of bradykinin, a potent vasodilator, from HMWK
Contact Activation Pathway
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- Involves tissue factor, factors VII and X, and Ca2+ and results in the production of factor Xa
- Initiated at the site of tissue injury with the exposure of tissue factor on activated endothelial cells and monocytes
- Tissue factor interacts with and activates factor VII, a circulating Gla-containing glycoprotein synthesized in the liver
Tissue Factor Pathway
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- Major physiologic inhibitor of coagulation
- Circulates in the blood associated with lipoproteins
- Directly inhibits factor Xa by binding to the enzyme near its active site
- Factor Xa-TFPI complex then inhibits the factor VIIa-tissue factor complex
Tissue Factor Pathway Inhibitor (TFPI)
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Activation of Prothrombin to Thrombin
- The common point in both pathways is the activation of factor X to factor Xa
- Factor Xa activates prothrombin (factor II) to thrombin (factor IIa)
- Thrombin, in turn, converts fibrinogen to fibrin
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- Single-chain glycoprotein synthesized in the liver
- The amino terminal region of prothrombin contains ten Gla residues, and the serine-dependent active protease site is in the carboxyl terminal region of the molecule
- Upon binding to the complex of factors Va and Xa on the platelet membrane, prothrombin is cleaved by factor Xa
PROTHROMBIN
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Control of Thrombin Levels
- The activation of thrombin is also regulated by specific thrombin inhibitors
- Antithrombin III is the most important since it can also inhibit the activities of factors IXa, Xa, XIa and XIIa.
- The activity of antithrombin III is potentiated in the presence of heparin
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Heparin can be antagonized by strongly cationic polypeptides such as __, which bind strongly to heparin, thus inhibiting its binding to antithrombin
protamine
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- derived from enzymatic or chemical cleavage of unfractionated heparin, have an advantage over heparin because:
1. They can be administered subcutaneously at home
2. They have greater bioavailability
3. They do not need frequent laboratory monitoring of bleeding parameters
Low molecular weight heparins (LMWHs)
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Genetic deficiency of either __ can cause venous thrombosis
protein C or protein S
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Patients with __ (which has a glutamine residue in place of an arginine at position 506) have an increased risk of venous thrombotic disease because factor V Leiden is resistant to inactivation by Activated Protein C (APC Resistance)
factor V Leiden
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Dissolution of Fibrin Clots
- Degradation of fibrin clots is the function of Plasmin, a serine protease that circulates as the inactive proenzyme, plasminogen
- Any free circulating plasmin is rapidly inhibited by 2-antiplasmin
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- binds to both fibrinogen and fibrin, thereby being incorporated into a clot as it is formed
Plasminogen
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__ and, to a lesser degree, urokinase are serine proteases which convert plasminogen to plasmin
Tissue plasminogen activator (tPA)
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- is produced as the precursor, prourokinase by epithelial cells lining excretory ducts
- activate the dissolution of fibrin clots that may be deposited in these ducts
- Following the release of plasminogen and plasmin they are rapidly inactivated by their respective inhibitors
Urokinase
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- Classic hemophilia
- It is an X-linked disorder resulting from a deficiency in factor VIII
- Individuals with deficiencies in factor VIII suffer joint and muscle hemorrhage, easy bruising and prolonged bleeding from wounds
- Treatment is accomplished by infusion of factor VIII concentrates prepared from either human plasma or by recombinant DNA technology
Hemophilia A
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- Deficiencies in factor IX
- Christmas disease
- X-linked recessive
Hemophilia B
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- Due to inherited deficiency in von Willebrand factor (vWF)
- The most common inherited bleeding disorder of humans
- Deficiency of vWF results in defective platelet adhesion and causes a secondary deficiency in factor VIII
von Willebrand Disease
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Factor XI and Contact Activation
- Deficiency in factor XI confers an injury-related bleeding tendency
- Originally termed hemophilia C
- Common in Ashkenazic Jews
- Inherited as an autosomal disorder with either homozygosity or compound heterozygosity
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- Seen in approximately 2% of patients with venous thromboembolic disease
- Autosomal dominant
- Mutations that affect synthesis or stability of antithrombin or from mutations that affect the protease and/or heparin binding sites of antithrombin.
- Clinical manifestations include deep vein thrombosis and pulmonary embolism
Antithrombin Deficiency
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- Measures the intrinsic pathway (which includes factors I, II, V, VIII, IX, X, XI, and XII) and the common pathway
- It shows possible coagulation factor deficiency as in hemophilia A & B
- The test is a used to assess the overall competence of the coagulation system, as a routine test to monitor heparin therapy, and as a preoperative screen for bleeding tendencies
- Prolonged time suggests the presence of warfarin, the existence of vitamin K deficiency or liver dysfunction
Activated Partial Thromboplastin Time (APTT)
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- Measures the rate of a patient's clot formation compared to a normal plasma control
- The plasma is first depleted of platelets, and a standard amount of thrombin added
- The test is used in the diagnosis of disseminated intravascular coagulation (DIC), and other conditions that can affect fibrinogen level such as liver disease
Thrombin Clotting Time Test
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- Tests the interaction between the platelets and the vessel walls
- Not useful as a screening test as it has a high false positive result, but is sometimes useful in von Willebrand's Disease and the effect of drugs on the coagulation system
Bleeding time
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- D dimer is a fibrin degradation product
- Used in the diagnosis of deep vein thrombosis
- High concentrations also occur in malignancy and pregnancy
D-dimer test
