Hemostasis and Thrombosis


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      Hemostasis (blood coagulation; blood clotting) is the physiological process that stops an hemorrage and forms the semi-solid blood clot, which is then reabsorbed in the course of several days, in parallel with tissue repair. Hemostasis is actually costituted by two independent and very different processes: platelet aggregation and fibrin precipitation. Platelet aggregation is faster but the platelet plug is short lived; fibrin precipitation is slower but yields the much longer lived fibrin clot. As any other phyiological process, blood coagulation may go wrong because of many reasons and several pathologies related to abnormal coagulation are known.

      Platelet activation. Platelets (thrombocytes) are fragments of the cytoplasm of the megakaryocytes of the bone marrow. They number >140.000/mm3 in the blood of healthy individuals, and are subject to rapid turnover (average lifespan is approx. 7 days). If platelet enter in contact with the collagen or the von Willebrand factor (a protein produced by the endothelium) because of lesions of the endothelial wall, they become activated and acquire the abiility to adhere to the tissue or to each other, thus forming the platelet plug over the lesion(s). Platelets are also activated by thrombin. Activated platelets produce and secrete local hormones called thromboxanes (whose precursor is arachidonic acid), that in turn activate and recruit other platelets, thus amplifying the reaction. Several drugs interfere with platelet activation: e.g. aspirin is an inhibitor of the enzyme cyclooxygenase that starts the pathway converting arachidonic acid to thromboxane.
      The process of platelet activation is relevant also to fibrin clotting: this is because activated platelets release coagulation factor V that participates to the activation of thrombin (coagulation factor V is present in the blood in two fractions: a circulating one and one contained in the platelets alpha-granules; it is a protein devoid of protease activity but required for the interaction between prothrombin and factor Xa).

      Fibrin clotting. Blood plasma contains 200-400 mg/dL of fibrinogen, a large (MW 340 KDa) soluble protein prduced by the liver. Proteolytic digestion of two short segments (fibrinopeptides) of its polypeptide chain at the carboxy- and amino-terminus converts fibrinogen into fibrin. Fibrin is essentially insoluble and readily aggregates forming a network that adheres to the platelet plug. The fibrin network is further stabilized by the formation of covalent isopeptide bonds catalyzed by coagulation factor XIII.
      The conversion of fibrinogen into fibrin is due to the specific proteolytic enzyme thrombin. The enzymes of the coagulation cascade, including thrombin, are peculiar and very substrate-specidic proteases whose surface glutamic acid residues are post-translationally carboxylated and converted to gamma-carboxy glutamates; they require calcium to function. The enzyme that gamma-carboxylates glutamic acid uses vitamin K as a cofactor and in avitaminosis K (a rare condition, being this vitamin produced by the intestinal flora) thrombin and the other coagulation enzymes are produced but are not post-translationally modified and are inactive. Hence deficit of vitamin K (observed during long term antibiotic treatments) or administration of its inhibitors (antivitamin K; e.g. warfarin) cause defective blood clotting.

      Thrombin (a Ser-protease) is present in blood as its inactive precursor prothrombin in which a C-terminal arm folds over the active site of the enzyme, and prevents binding of fibrinogen. Proteolytic digestion of the C-terminal arm by coagulation factors X and V (themselves Ser proteases present in the blood as inactive precursors) is required to initiate the process.
      The activation of factors X and V requires a proteolytic cascade in which inactive proteases precursors are activated and digest their specific substrate. The proteolytic cascade has two functions: (i) signal amplification because each activated enzyme activates many substrates; and (ii) control because specific or generic inhibitors can block the process at different points. Two quasi-independent proteolytic cascades can activate prothrombin: the intrinsic and extrinsic pathways.
      The intrinsic pathway is activated by the plasma protein High Molecular Weight kininogen (HMWk), which is activated by contact with collagen. This pathway is longer and includes coagulation factors XII, XI, X, IX, VIII. It is tested by measuring the thromboplastin time.
      The extrinsic pathway is activated by the tissue factor, which resides in the tunica media of the vessels and would not normally contact blood. This pathway is shorter and includes coagulation factors VII, and X. It is tested by measuring the prothrombin time.

von Willebrand factor and von Willebrand disease
      In 1926 the finnish physician Erik von Willebrand described a complex and highly variable autosomal inheritable disease of coagulation, rightly named after him. von Willebrand disease is due to genetic mutations of von Willebrand factor a large multimeric protein produced by megakaryocytes and endotelial cells, which participates to various coagulation processes. von Willebrand factor is not an enzyme; rather it is a protein capable of binding several other proteins, causing their aggregation. In particular under shear stress (i.e. where the blood flow is turbulent) vWF binds to collagen and to platelets, and favors the formation of the platelet plug, thus participating to the first process of coagulation. Moreover, vWF binds to coagulation factor VIII and stabilizes it against unfolding, prolonging its active half-life. This causes the second process of coagulation (i.e. conversion of fibrinogen to fibrin) to be more active in the region of the vascular lesion and the platelet plug. The amount of vVF in the serum can be measured by means of immunoassays; moreover the vVF gene on chromosome 12 can be sequenced if vW disease is suspected, because of a hemorragic syndrome affecting both phases of coagulation.

TABLE 1: A list of the main coagulation components and the pertinent laboratory tests (due to faulty identifications, not all numbers are used)
Component Definition and properties Laboratory test
Components required for the formation of the platelet plug
Platelets fragments of the megakaryocyte cytoplasm platelet count (normal value >150,000 mm3); platelet aggregation test
von Willebrand factor protein present on the vessel wall and in the plasma vW antigen (measures total vW factor concentration by electroimmunoassay); vW multimer composition
Factors required for the formation of the fibrin network
Factor I fibrinogen (plasma)
Factor II prothrombin (plasma)
Factor V proaccelerin, labile factor (plasma) specific activity test (measurement of the ability of the patient's plasma to complement the coagulation of a factor V deficient test plasma)
Factor VII proconvertin, stabile factor (plasma) specific activity test (same as in the case of factor V but test plasma lacks factor VII)
Factor VIII antihemophylic factor (plasma) specific activity test (same as in the case of factor V but test plasma lacks factor VIII)
Factor IX Christmas factor, plasma thromboplastin component (plasma) specific activity test (same as in the case of factor V but test plasma lacks factor IX)
Factor X Stuart factor (plasma) specific activity test (same as in the case of factor V but test plasma lacks factor X)
Factor XI Plasma Thromboplastin Antecedent, PTA (plasma) specific activity test (same as in the case of factor V but test plasma lacks factor XI)
Flechter factor prekallicrein (plasma)
Fitzgerald factor high molecular weight kininogen (plasma)
Factor XII Hageman factor (plasma) specific activity test (same as in the case of factor V but test plasma lacks factor XII)
Factor XIII fibrin stabilizing factor (plasma) measurement of clot stability upon 24 hours incubation in 5 M urea
Tissue Thromboplastin cell surface lipoprotein
Platelet factor 3 a phospholipid from the platelet membrane
Thrombomodulin membrane protein of endothelial cells
Intrinsic pathway fibrinogen, prothrombin, factors V, VIII, IX, X, XI, XIII, prekallikrein, HMW kininogen Partial Thromboplastin Time (PTT)
Extrinsic pathway fibrinogen, prothrombin, factors V, VII, X Prothrombin Time (PT)
Factors that control coagulation and digest the fibrin network
Plasminogen activated by a tissue specific protease to plasmin, digests the fibrin network Enzymatic activity assay (after activation)
alpha2 antiplasmin
antithrombin III Specific inhibitor of thrombin Measured by immunoassay or by its ability to inhibit the enzymatic activity of thrombin
Protein C (plasma) Inhibitor of factors VIIa and Va Electroimmunoassay
Protein S (plasma) Required for the function of Protein C Electroimmunoassay

      Hemorragic disorders may be due to three groups of causes, usually quite clearly distinguishable on clinical grounds: (i) defects of the vascular wall (e.g. scurvy, allergic purpura, hereditary hemorragic teleangiectasia, etc.); these are not disorders of the coagulation and all laboratory tests of coagulation are normal. (ii) Platelets disorders, due either to a reduction in number or to functional impairment; the cardinal manifestations of these disorders are prolonged bleeding and purpura, i.e. formation of cutaneous (and internal) petechiae on minor traumas. (iii) Defects of fibrin clotting (most often due to hereditary genetic defects of coagulation factors); the cardinal manifestation is temporary hemostasis (because of the formation of the platelet plug) followed by multiple episodes of re-opening of the lesion and bleeding (because of the dissolution of the platelet plug before any fibrin clot is formed).

      Some important hemorragic syndromes
      Hemophilias are inherited, X-linked disorders in which one coagulation factor is not produced or produced in an inactive form. Two main variants are described: hemophilia A in which coagulation factor VIII is affected and hemophilia B in which coagulation factor IX is affected, the disease is sex-linked and recessive: only males are affected (except for the very rare cases of homozygous females). Very rare instances of acquired hemophilias are described, due to an autoimmune response against factors VIII or IX. Wounds, even minor ones, undergo temporary sealing because of the formation of the platelet plug, but they re-open and bleed repeatedly because the fibrin clot is not formed. Some anatomical districts are specially prone to hemorrages, most notably the joints. Joints may experience frequent, even though very minor traumas (e.g. the knee and ankle, on which the body weight rests), which usually go unnoticed. In hemophilic patients these produce massive, painful internal hemorrages (hemartros), not to be confused with atrthritis.
      Laboratory diagnosis: the bleeding pattern and familial anamnesis should raise suspicion, which is confirmed by complementation tests and/or gene sequencing. Therapy is via transfusion of coagulation factors obtained from donated blood. Recombinant production of coagulation factors in bacteria is not a viable solution because bacteria do not carry out the necessary post-translational modifications (glutamic carboxylation).

      Excess anticoagulant therapy. Anticoagulant therapy is a common clinical practice that reduces the risk of thrombosis and infarctions in patients at risk of such diseases. It is usually carried out using inactive analogues of vitamin K (antivitamins) that block the enzyme responsible for the carboxylation of glutamate residues; the coagulation factors produced are partially inactivated. In case of overdosage (in children; in patients who are confused), the inactivation of coagulation factors may be massive and may produce a syndrome similar to hemophilia.

      Von Willebrand disease is a mixed-type coagulation defect that affects both the platelet- and fibrin-dependent processes; see the description given above.

      Afibrinogenemia is a rare genetic disorder, resembling hemophilias; however the inheritance is autosomal and recessive (the gene of fibrinogen is located on chromosome 4), thus both males and females may be affected. Laboratory diagnosis is by gene sequencing.

      Disseminated intravascular coagulation (DIC) occurs because of an abnormal activation of thrombin and fibrinogen, due to the presence of proteases in the serum. The causes may be varied: acute pancreatitis (with resorption of trypsin and chymotrypsin); sepsis due to coagulase-producing bacteria; snake bite (some snake venoms contain coagulase enzymes); etc. This is a severe, potentially letal condition, in which thromby and microthrombi form in the veins and may cause embolism. The defect of coagulation is due to the consupmtion of fibrinogen, is clinically similar to afibrinogenemia, and causes high risk of internal and external hemorrages. The blood serum contains several protease inhibitors that should protect against DIC; howeverthese may be overcome if the amount of anomalous proteases in blood is high.

      Advanced stages of chronic liver failure. Coagulation factors are produced by the liver, and in late stages of liver failure (e.g. due to cirrhosis, chronic viral hepatitis, liver cancer, etc.) their bisynthesis may be impaired, resulting in coagulation defects clinically similar to hemophilias (notice that the platelets are produced by megakariocytes in the bone marrow, and may result normal).

      Fibrinolysis, the dissolution of the fibrin newtork, is an important physiological event, and is due to selective proteolysis. The enzyme responsible for this process is called plasmin and is present in the blood as the inactive precursor plasminogen. Plasminogen is activated by thrombin, thus the coagulation process is auto-regulated.

      Thrombophilias are congenital, inherited defects of coagulation that may be due to variants of coagulation factors more prone to autoactivation or to defects of plasminogen. They cause an increased risk of thrombosis and infarctions.

Type of abnormality Pathogenesis Test(s) for diagnosis
Purpuras (insufficient platelet functioning) Idiopathic Thrombocytopenic Purpura Low platelet count, in the absence of bone marrow disease (possibly autoimmune cause?)
  Secondary Thrombocytopenias Low platelet count, in the presence of bone marrow diseases (e.g. aplasia, leukemia) or accelerated platelet turnover (e.g. allergic, drug induced)
  Glanzmann's Thromboasthenia Normal platelet count, but reduced tendency of the platelets to aggregate (the disease is inherited as an autosomal recessive trait)
  von Willebrand disease Normal platelet count, hereditary deficiency (autosomal, dominant) of the von Willebrand factor, required for the adhesion of platelets to the damaged endothelium
Hemorragic syndromes (impaired coagulation) Hereditary deficiency of factor VIII (Hemophylia A) Bleeding and hemorrages; heredity (X-linked, recessive); gene sequencing (the locus of this gene resides in the X chromosome); increased thromboplastin time with normal prothrombin time
  Hereditary deficiency of factor IX (Hemophylia B) Bleeding and hemorrages; heredity (X-linked, recessive); gene sequencing (the locus of this gene resides in the X chromosome); increased thromboplastin time with normal prothrombin time
  Hereditary deficiency of fibrinogen Reduced fibrinogen in the plasma associated to mild hemorrages (DIC and hemodilution should be excluded); increased thromboplastin and prothrombin times
  Exhaustion due to Disseminated Intravascular Coagulation (DIC) DIC is an abnormal activation of coagulation due to release of digestive enzymes in the blood (during acute pancreatitis), or to release of the tissue factor of coagulation (e.g. in leukemias and other cancers; in massive traumas), or to coagulase toxins (e.g. snake venom, gram negative sepsis)
Increased coagulation and thrombosis Disseminated Intravascular Coagulation (DIC) see above
  Flebitis Damage of the vascular wall releases or exposes the tissue factor of coagulation
  Impaired circulation in the atria Blood remaining in the heart atria because of atrial fibrillation or stenosis of the atriventricular valve is at risk of intravascular coagulation because of poor mixing and reduced activation of plasmin
  Hereditary deficiency of plasminogen A rare autosomally inherited disorder associated to DIC and to "ligneous" conjunctivitis (organization of conjunctival microhemorrages)
  Hereditary variants of factor V Some hyperfunctional variants of factor V (e.g. factor V Leiden) may cause hypercoagulation and tendency to thrombosis (thrombophilia)

      Laboratory tests of coagulation. The most important test of coagulation is the prothrombin time (PT). Blood is drawn an stored in a tube containing sodium citrate as an anticoagulant (it is important to fill the test tube to the exact volume required since in this test the blood:anticoagulant ratio must be standardized). Citrate prevents coagulation by chelating calcium ions. In the clinical laboratory the plasma is prepared and calcium chloride is added in order to saturate citrate and obtaining a standard concentration of the free calcium ion. Tissue factor is also added and the time required for the precipitation of fibrin is measured by turbidimetry. Typical prothrombin time in healthy adults is in the order of 12-14 sec.
      Prothrombin time is increased in the following conditions: administration of warfarin or deficiency of vitamin K; Disseminated Intravascular Coagulation syndrome; liver failure (the liver produces all the coagulation factors); congenital afibrinogenemia; hereditary deficiency of coagulation factors V and X. Prothrombin time is normal in platelet diseases and in hereditary coagulation disorders affecting the intrinsic pathway.
      The International Normalized Ratio (INR) is the ratio between the patient's PT and the average PT of healthy subjects as measured by the same laboratory. In practice the INR measures by how much the patient's PT is increased (or decreased) with respect to the controls:
INR = patient's PT / control PT
The function of INR is to provide a parameter with lower inter-laboratory variability; thus its use is now recommended over the PT. INR is particularly important in the follow-up of anticoagulant therapy. Normal value of INR is 1 (the patient's PT equals the average value of healthy subjects); an increase to 2-3 (double or triple the average value) is typical of an effective anticoagulant therapy, and is considered a moderate increase; an increase to 5 or more indicates risk of internal hemorrages, and in the absence of inherited coagulation defects or severe liver failure is indicative of excess anticoagulant therapy or accidental intoxication with anticoagulant drugs.
      The partial thromboplastin time (PTT) is a clinical measurement similar to the prothrombin time, in which the intrinsic pathway is activated by addition of phospholipids and calcium to the patient's plasma. Values in the healthy adult range between 30 and 50 sec. The thromboplastin time is increased in the following conditions administration of warfarin or deficiency of vitamin K; Disseminated Intravascular Coagulation syndrome; liver failure (the liver produces all the coagulation factors); congenital afibrinogenemia; hereditary deficiency of coagulation factors VIII and IX (hemophylias A and B).

Clinical interpretation of abnormal coagulation times
PT /INRPTTclinical interpretation
increasednormalLiver disease, vitamin K insufficiency, decreased or defective factor VII, chronic low-grade disseminated intravascular coagulation (DIC), anticoagulation drug (warfarin) therapy
normalincreasedDecreased or defective factor VIII, IX, XI, or XII, von Willebrand disease (severe type), presence of lupus anticoagulant, autoantibody against a specific factor (e.g., factor VIII)
increasedincreasedDecreased or defective factor I, II, V or X, severe liver disease, acute DIC, warfarin overdose

      Clinical examples
      1) A male child of four presents abdominal pain, blood in the feces, prolonged bleeding from occasional minor wounds since a week. A blood test reveals:
Hemoglobin 10 g/dL (normal range 13-15 g/dL)
Erythrocyte count 3 x 106/μL; (normal value 4-5 x 106/μL;)
White cell count 5 x 103/μL; (normal value 4-9 x 103/μL;)
Platelet count 2.2 x 105/μL; (normal value 1.5-4 x 105/μL;)
PT 90 sec (normal value 12-14 sec)
INR 8 (mormal value 1)
PTT 130 sec (normal value 25-35 sec)
Other analytes tested were unremarkable.
Analysis of the case: the child has an hemorragic syndrome. Platelet count is within the normal range, thus the platelet phase of coagulation is probably unaffected; besides the clinical presentation is not typical for a disease of platelets.
PT, INR, and PTT are all significantly increased, thus the disease affects the fibrin clot formation phase. Anemia is present, but it is most probably a consequence of hemorrages.
Possible diagnoses are: Von Willebrandt disease; an ereditary defect of coagulation; warfarin poisoning (did the child find and ingest an anticoagulant medication? is anybody in the family under anticoagulant therapy? did the child ingest rat poison?); DIC. Inherited coagulopathies are a less likely hypothesis because of two reasons: (i) the disease's onset can be traced in time a week ago (inherited defects of coagulation are present at birth and usually manifest themselves earlier); and (ii) both PT and PTT are affected. However excluding inherited coagulopathies on these grounds would be hazardous. Liver insufficiency is an unlikely hypothesis given that other blood parameters (bilirubin, liver enzymes) are unremarkable.
How to proceed. Several further analyses are required to establish the diagnosis: (i) measure the serum concentration of coumarin derivatives (warfarin and related anticoagulant drugs) to establish whether anticoagulant poisoning has occurred; (ii) sequence the genes of coagulation factors, including Von Willebrandt's; (iii) measure the serum fibrinogen concentration (reduced in DIC).
After the above analysis were carried out a diagnosis of anticoagulant intoxication could be made; it turned out that the child had found and ingested some anticoagulant pills destined to his grand-father.

      Thrombosis is the pathological process of intravascular coagulation. It is usually due to the activation of the extrinsic coagulation pathway in the proximity of a lesion of the vascular endothelium, e.g. over an atheromatous plaque in an artery or in an inflamed vein (thromboflebitis). Arterial thrombi rarely grow to form large structures because of the rapid blood flow; however they may cause infarctions or may become organized (i.e. they may be transformed into scars, rigid because of the deposition of collagen) and cause a permanent stenosis of the affected artery. Venous thrombi are large and fragile structures and may release emboli that may in turn cause arterial obstructions and infarctions.
      A very severe condition occurs if the thrombus forms in the left (or more rarely in the right) atrium of the heart, often during chronic atrial fibrillation. A fragment of the thrombus may be expelled in the pulmonary artery and cause massive pulmonary embolism, a condition that is often fatal.
      Whenever a risk condition is present, anticoagulant therapy is indicated. Unfortunately risk conditions may not be easy to detect (e.g. flebitis of internal veins).

Questions and exercises:
1) The laboratory test that probes the intrinsic coagulation pathway is:
Thromboplastin time
Prothrombin time
Platelet aggregation test

2) The normal platelet count in a healthy adult is:
140,000-400,000 /mm3
140,000-400,000 / dL
140,000-400,000 / mL

3) The inheritance mechanism of classical hemophylias is:
autosomal and recessive
sex-linked and recessive
sex-linked and dominant

4) A patient presents multiple petechiae, and its laboratory analyses reveal a reduced platelet count. This clinical picture may be represent the early stage of:
chronic liver failure

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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).

is it possible to take gulonolactone oxidase to synthesize vitamin C
instead of vitamin C supplement?
Bellelli: no, this approach does not work. The main reason is that
the biosynthesis of vitamin C, as almost all other metabolic processes, occurs intracellularly.
If you administer the enzyme it will at most reach the extracellular fluid but will not be
transported inside the cells to any significant extent. Besides, there are other problems
in this type of therapy (e.g. the enzyme if administered orally, may be degraded by digestive
proteases; if administered parenterally, may cause the immune system to react against a
non-self protein). In theory one could think of a genetic modification of the inactive human
gene of gulonolactone oxidase, but the risk and cost of this intervention would not be
justified. In addition to these considerations, except for cases of shipwreckage or
other catastrophes, a proper diet or administration of tablets of vitamin C is effective,
risk-free and unexpensive, thus no alternative therapy is reasonable. However, I express my
congratulations for your search on the biosynthesis pathway of ascorbic acid.

Resorption and not reabsorption would lead to hypercalcemia ie bone matrix being broken down.
Bellelli: I am not sure to interpret your question correctly. Resorption indicates destruction of the bone matrix and release of calcium and
phosphate in the blood, thus it causes an increase of calcemia. Reabsorption usually means active transport of calcium from the renal tubuli to the blood, thus
it prevents calcium loss. It prevents hypocalcemia, and thus complement bone resorption. To avoid confusion it is better use the terms "bone resorption" and "
renal reabsorption of calcium". If you have a defect in renal reabsorption, parthyroid hormone will be released to maintain a normal calcium level by means of
bone resorption; the drawback is osteoporosis.

In Reed and Frost model: I haven't understood what is the relationship
between K and R reproductive index. Thank you Professor!
Bellelli: in the Reed and Frost model K is the theoretical upper limit of
R0. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R0 is the value of R one measures at the beginning
of the epidemics, when in principle all the population is susceptible.

What is the link between nucleotide metabolism and immunodeficiencies and mental retardation?
Bellelli: the links may be quite complex, but the principal ones are as follows:
1) the immune response requires a replication burst of granulocytes and lymphocytes, which in turn requires
a sudden increase of nucleotide production, necessary for DNA replication. Defects of nucleotide metabolism
impair this phase of the immune defense. Notice that the mechanism is similar to the one responsible of
anemia which requires a sustained biosynthesis of nucleotides at a constant rate, rather than in a burst.
2) Mental retardation is mainly due to the accumulation of nulceotide precursors in the brain of the
newborn, due to the incompletely competent blood-brain barrier.

How can ornithine transaminase defects cause hyperammonemia? Is it due to the accumulation
of ornithine that blocks the urea cycle or for other reasons?
Bellelli: ornithine transaminase is required for the reversible interconversion of ornithine
and proline, and thus participates to both the biosynthesis and degradation of ornithine. The enzyme is
synthesized in the cytoplasm and imported in the mitochondrion. Depending on the metabolic conditions
the deficiency of this enzyme may cause both excess (when degradation would be necessary) or defect
(when biosynthesis would be necessary) of ornithine; in the latter case, the urea cycle slows down. Thus
there is the paradoxical condition in which alternation may occur between episodes of hyperammonemia
and of hyperornithinemia.

When we use the Berthelot's reaction to measure BUN do we also have to
measure the concentration of free ammonia before adding urease?
Bellelli: yes, in principle you should. Berthelot's reaction detects ammonia,
thus one should take two identical volumes of serum, use one to measure free ammonia,
the other to add urease and measure free ammonia plus ammonia released by urea. BUN is
obtained by difference. However, free ammonia in our blood is so much lower than urea that
you may omit the first sample, if you only want to measure BUN.

Why do we have abnormal electrolytes in hematological neoplasia e.g.
Bellelli: I do not have a good explanation for this effect, which may have
multiple causes. However, you should consider two factors: (i) acute leukemias cause a massive
proliferation of leukocytes (or lymphocytes depending on the cell type affected) with a very
shortened lifetime; thus you observe an excess death rate of the neoplastic cells. The dying
cells release in the bloodstream their content, which has an electrolyte composition different
from that of plasma: the cell cytoplasm is rich in K and poor in Na, thus causing hyperkalemia.
(ii) the kidney may be affected by the accumulation of neoplastic white cells or their lytic products.

Gaussian curve: If it is bimodal is it more likely to be a "certain diagnosis" than if it is
unimodal or does it only show the distinguishment from health?
Bellelli an obviously bimodal Gaussian curve indicates that the disease is clearly
separated from health: usually it is a matter of how precise and clear-cut is the definition of the disease.
For example tuberculosis is the disease caused by M. tuberculosis, thus if the culture of the sputum is
positive for this bacterium you have a "certain" diagnosis (caution: the patient may suffer of two diseases,
e.g. tuberculosis and COPD diagnosis of the first does not exclude the second). However, in order to have
a "certain" diagnosis it is not enough that distribution of the parameter is bimodal, it is also required that the
patient's parameter is out of the range of the healthy condition: this is because a distribution can be
bimodal even though it is composed by two Gaussians that present a large overlap, and the patient's
parameter may fall in the overlapping region. Thus, in order to obtain a "certain" diagnosis you need to
consider not only the distribution of the parameter(s) but also the patient's values and the extent of the
overlapping region.

Prof can you please elaborate a bit more on the interhuman variability and its difference
with the interpopulation variability please?
Bellelli: every individual is a unique combination of different alleles of the same genes;
this is the source of interindividual variability. Every population is a group of individuals who intermarry and
share the same gene pool (better: allele pool). Every allele in a population has its own frequency. Two
population may differ because of the diffferent frequencies of the same alleles; in some cases one
population may completely lack some alleles. The number and frequencies of alleles of each gene
determine the variance. If you take two populations and calculate the cumulative interindividual variance
of the population the number you obtain is the sum of two contributions: the interindividual variance within each population, plus the interpopulation variance
between the means of the allele frequencies. For example, there are human population in which the frequency of blood group B is close to 0% and other populati
ons in which it is 30% or more.

Prof can you please explain again the graph you have showed us in class about thromboplastin?
(Y axis=abs X axis= time)
Bellelli: the graph that I crudely sketched in class represented the signal
of the instrument (an absorbance spectrophotometer) used to record the turbidity of the
sample (turbidimetry). The plasma is more or less transparent, before coagulation starts.
When calcium and the tissue factor (or collagen) are added. thrombin is activated and begins
digesting fibrinogen to fibrin; then fibrin aggregates. The macroscopic fibrin aggregates cause
the sample to become turbid, which means it scatters the incident light. The instrument reads
this as a decrease of transmitted light ( an increase of the apparent absorbance) and the
time profile of the signal presents an initial lag phase, which is called the protrombin or
thromboplastin time depending on the component which was added to start coagulation
(tissue factor or collagen).

Prof can you please explain the concept you have described in class about
the simultaneous hypercoagulation and hemorrhagic syndrome? How can this occur?
Bellelli: The condition you describe is observed only in the Disseminated
Intravascular Coagulation syndrome. Suppose that the patient experiences an episode of
acute pancreatitis: tripsin and chymotripsin are reabsorbed in the blood and proteolytically
activate coagulation causing an extensive consumption of fibrinogen and other coagulation
factors. Tripsin and chymotripsin also damage the vessel walls and may cause internal
hemorrages, but at that point the consumption of fibrinogen may have been so massive that
not enough is left to form the clot where the vessel has been damaged, causing an internal
hemorrage. Pancreatitis is a very severe, potentially lethal condition, and DIC is only one of
the reasons of its severity.

You said that certain drugs (ethanol, cocaine, cannabis, opiates...) cause a
necessity of higher and higher dosage, for two reasons: the enzyme in the liver is inducible and
the receptors in the brain are expressed less and less. So, first, I am not sure I got it right, and
second I did not understand how expressing less receptors leads to a necessity of higher
Bellelli: You got it correctly, but the detailed mechanism of resistance may
vary among different substances, and not all drugs cause adaptation.
The reason why reducing the number of receptors may require an increased dosage of the drug
is as follows: suppose that a certain cell has 10,000 receptors for a drug. When bound to its
agonist/effector, each receptor produces an intracellular second messenger. Suppose that in
order for the cell to respond 1,000 receptors must be activated. The concentration of the
effector required is thus the concentration that produces 10% saturation. You can easily
calculate that this concentration is approximately 1/10 of the equilibrium dissociation constant
of the receptor-effector complex (its Kd), the law being
Fraction bound = [X] / ([X]+Kd)
where [X] is the concentration of the free drug.
After repeated administration, the subject becomes adapted to the drug, and his/her cells
express less receptors, say 5,000. The cell response will in any case require that 1,000
receptors are bound to the effector and activated, but this now represents 20% of the total
receptors, instead of 10%. The drug concentration required is now 1/4 of the Kd.
Continuing administration of the drug further reduces the cell receptors, but the absolute
number of activated receptors required to start the response is constant; thus the fewer
receptors on the cell membrane, the higher the fraction of activated receptors required.

Why does hyperosmolarity happen in type 2 diabetes and not in type 1?
Bellelli: Hyperosmolarity can occur also in type 1 diabetes, albeit
infrequently. The approximate formula for plasma osmolarity is reported in the lecture on
osmolarity = 2 x (Na+ + K+) + BUN/2.8 + glucose/18
this is expressed in the usual clinical laboratory units (mEq/L for electrolytes, g/dL for non-
electrolytes). The normal values are:
osmolarity = 2 x (135 + 5) + 15/2.8 + 100/18 = 280 + 5.4 + 5.6 = 291 mOsmol/L
Let's imagine a diabetic patient having normal values for electrolytes and BUN, and glycemia=400 mg/dL:
osmolarity = 280 + 5.4 + 22.4 = 307.8 mOsmol/L
The hyperosmolarity in diabetes is mainly due to hyperglycemia, even though other factors
may contribute (e.g. diabetic nefropathy); however the contribution of glucose to osmolarity is
relatively small. As a consequence in order to observe hyperosmolarity the hyperglycemia
should be extremely high; this is more often observed in type 2 than in type 1 diabetes, for
several reasons, the most relevant of which is that in type 1 diabetes all cells are starved of
glucose, and the global reserve of glycogen in the body is impoverished: there is too much
glucose in the blood and too few everywhere else, thus reducing, but not abolishing, the risk of
extreme hyperglycemia. Usually in type 2 diabetes the glycogen reserve in the organism is not
impoverished, thus the risk of extreme hyperglycemia is higher.

Hemostasis and Thrombosis lecture: I don't understand why is sodium citrate
added to the serum solution to measure the prothrombin time.
Bellelli: in order to measure PT or PTT you want to be able to start the
coagulation process at an arbitrary time zero, and measure the increase in turbidity of the
serum sample. To do so you need (i) to prevent spontaneous coagulation with an anticoagulant;
and (ii) to be able to overcome the anticoagulant at your will. Citrate (or oxaloacetate; or EDTA)
has the required characteristics: it chelates calcium, and in this way it prevents coagulation;
but you can revert its effect at your will by adding CaCl2 in excess to the amount
of citrate. You cannot obtain the same effect with other anticoagulants (e.g. heparin) whose
action cannot be easily overcome.

Dear professor I cannot do the self evaluation test because it says the the
time has expired It is not possible because I havent even started them
Bellelli: this is due to the fact that the program registers your name and
matricola number from previous attempts. I shall fix this bug. Meanwhile try to use a fake
matricola number.

How is nephrotic syndrome associated hypoalbuminemia as you described
in methods of analysis of protein because seems counterintuitive
Bellelli: nephrotic syndrome is an autoimmune disease in which the
glomerulus is damaged and the filtration barrier is disrupted; diuresis is normal but there is
loss of proteins (mostly albumin) in the urine.
I m sorry i confused polyurea with hypoalbuminemia but my question still
stands during glomerulonephritis you mentioned something of polyurea as compensation
i could not follow how this compensation mechanism works and collapse after some time in
Bellelli: the condition you describe is NOT characteristic of acute
glomerulonephritis. In glomerulonephritis there is damage of the glomerulus and severely
impaired GFR. Thus the diuresis is severely reduced, and due to impaired filtration proteins
appear in the urine.
The condition you describe corresponds to the initial stage of chronic kidney failure,
usually due to atherosclerosis, diabetes, hypertension or other type of damage of the kidney
tissue. In this case GFR is impaired, albeit to a lesser extent than in glomerulonephritis, and the
excretion of urea is reduced. This leads to increased BUN. However the increased concentration
of urea reduces the ability of the tubuli to reabsorb water, because of osmotic reasons, yielding
compensatory polyuria. The patient has reduced GFR but normal or increased diuresis (urine
volume in 24 hours). To some extent this effect is beneficial, as it favors the elimination of
urea; however it cannot completely solve the problem and in any case the progression of the
disease leads to kidney insufficiency. In its essence the point is that a moderately reduced GFR
can be partially compensated by reduced tubular reabsorption; a severely reduced GFR cannot.

Lecture on Hemogas analysis interpretation of complex cases standard pH
Why if PCO2 is less than 40 mmHg it is absorbed during equilibration? Thank you in advance
Bellelli: if PCO2 of the patient's blood sample is less than 40 mmHg, when
the machine equilibrates with 40 mmHg CO2 the gas is absorbed: i.e. the new PCO2 becomes
40 mmHg and the total CO2 of the sample increases; as CO2 is the acid of the buffer, the
standard pH (in this case) decreases, whereas standard bicarbonate will slightly increase.

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