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 (firbinopeptides) 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. Thrombin is a peculiar protein whose surface glutamic acid residues are post-translationally carboxylated and converted to gamma-carboxy glutamates; it requires 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 is produced but is not post-translationally modified and is 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.

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).

      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.

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

      Laboratory tests of coagulation. The most important test of coagulation is the prothrombin time. 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 thromboplastin time 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).


      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.

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