Thrombosis, thrombophilia and antithrombotic therapy

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Thrombosis, thrombophilia and antithrombotic
therapy

• By Carl Peters

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Definitions

• Thrombi solid masses or plugs formed in the
  circulation from blood constituents
• Both arterial and venous thrombosis can occur.
• Thrombophilia term used to describe the
  inherited or acquired disorders of the
  haemostatic mechanism which predispose to
  thrombosis.
• Hypofibrinolysis cant lyse clots normally.

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Pathogenesis

• Virchows triad suggests three components
  important in thrombus formation
• Slowing down of blood flow (venous)
• Hypercoagulability of the blood (venous)
• Vessel wall damage (arterial)

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Remember

• Risk stratification represents a spectrum
• Overall aim to decide
• What interventions can be implemented?
• Specifically, what anticoagulants to use?
• How long to use them?
• And, when to use them (e.g. surgery, pregnancy)?

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Arterial thrombosis
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Common locations

• Coronary arteries Þ myocardial infarction
• Carotid / vertebral / intracerebral arteries Þ
  ischaemic stroke
• Peripheral arteries (usually leg) Þ ischemia and
  (eventually) gangrene
• Mesenteric arteries Þ bowel infarction
• Retinal arteries Þ loss of vision
• Placental arteries Þ placental insufficiency,
  miscarriage, and major complications of pregnancy
  including eclampsia and pre-eclampsia, abruptio
  placentae, and intrauterine growth retardation.

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Pathogenesis

• Atherosclerosis of arterial wall, plaque rupture
  and endothelial injury expose blood to
  subendothelial collagen and tissue factor.

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Haemostasis pathway
Injury Collagen exposure
Tissue Factor Platelet adhesion
Coagulation Cascade Release reaction
Platelet
aggregation Fibrin
Primary haemostatic plug
Secondary haemostatic plug
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Coagulation cascade
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Pathogenesis cont

• Platelet deposition and thrombus formation are
  important in the pathogenesis of atherosclerosis
• Platelet-derived growth factor (PDGF) stimulates
  the migration and proliferation of smooth cells
  and fibroblasts in the arterial intima. Regrowth
  of endothelium and repair at the site of arterial
  damage and incorporated thrombus result in
  thickening of vessel wall.

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Risk factors (arterial)

• Related to the development of atherosclerosis
• Northwick Park heart study (Lancet. 1986 Sep
  62(8506)533-7) showed that elevated plasma
  levels of factor VII and fibrinogen are the
  strongest independent predictors of coronary
  events.

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Risk factors (arterial)

• Positive family history
• Male sex
• Hyperlipidaemia
• Hyperhomocysteinaemia
• Low serum folate, vitamin B12, vitamin B6
• Hypertension
• Diabetes mellitus
• Gout
• Polycythaemia
• Cigarette smoking
• ECG abnormalities
• Elevated factor VII and fibrinogen (elevations of
  one std. deviation associated with increases of
  risk of 62 and 84 respectively of IHD)
• Lupus anticoagulant
• Collagen vascular diseases
• Bechets disease (chronic, multisystem
  inflammatory disease)
• F VIII

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Hyperhomocysteinaemia

• Mild hyperhomocysteinemia in approx. 5 to 7
  percent of the general population
• Severe hyperhomocysteinemia is rare
• Patients with mild hyperhomocysteinemia are
  asymptomatic until the third or fourth decade of
  life when premature coronary artery disease may
  develop, as well as recurrent arterial and venous
  thrombosis

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Dietary protein
Remethylation
Trans-sulphuration
Folic acid
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Hyperhomocysteinaemia

• Homocyteine derived from dietary methionine.
• Removed by either remethylation or
  trans-sulphuration
• Classic homocystinuria
• rare autosomal dominant disorder
• caused by deficiency of cystathione
  beta-synthetase, enzyme responsible for
  trans-sulphuration
• Vascular disease and thrombosis major features of
  disease
• Heterozygous CBS deficiency
• present in around 0.5 of popn.
• leads to moderate increase in homocysteine.
• Methylene tetrahydrofolate reductase (MTHFR)
• involved in the remethylation pathway
• a common thermolabile variant may be responsible
  for mild homocysteinaemia, although may only be
  seen in presence of folate deficiency.
• Acquired risk factors for homocysteinaemia
  include deficiencies of folate, B12 or B6, drugs
  (e.g. cyclosporine), renal damage and smoking.
  Levels also increase with age and higher in men
  and post-menopausal women.
• NB also a risk factor for venous thrombosis

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Hyperhomocysteinaemia

• Diagnosis plasma homocysteine measured on a
  morning specimen after an overnight fast. 
  Because homocysteine is continuously released by
  blood cells, the specimen must be centrifuged and
  the plasma separated immediately to avoid falsely
  elevated values
• Elevations in plasma homocysteine are typically
  caused either by genetic defects in the enzymes
  involved in homocysteine metabolism or by
  nutritional deficiencies in vitamin cofactors
• Homocysteine gt13.1 umol/L- Folic acid 5 mg,
  Vitamin B6 100 mg, Vitamin B12, 2000 ug/day

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Venous thrombosis
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Common locations

• most often occurs in the leg, either in the
  superficial veins (superficial phlebitis), or in
  the deep veins (deep venous thrombosis)
• cerebral sinus
• retinal veins/and or arteries
• veins in the arms or upper thorax
• veins in the mesentery
• veins in bones, usually the hip or jaw, causing
  bone death (osteonecrosis)
• veins and/or arteries in the placenta, causing
  placental insufficiency, miscarriage, and major
  complications of pregnancy including eclampsia
  and pre-eclampsia, abruptio placentae, and
  intrauterine growth retardation.

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Pathogenesis

• Increased systemic coagulability and stasis are
  most important
• Vessel wall damage being less important than in
  arterial thrombosis, although may be important in
  patients with sepsis and in-dwelling catheters
• Stasis allows the completion of blood coagulation
  at the site of initiation of the thrombus (e.g.
  behind the valve pockets of the leg veins in
  immobile patients)

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Risk factors

• Related to coagulation abnormality
• Hereditary hemostatic disorders
• Factor V Leiden
• Prothrombin G20210A variant
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
• Abnormal fibrinogen
• Abnormal plasminogen

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Risk factors cont

• Acquired haemostatic disorders (some of these may
  also occur as inherited disorders)
• Raised plasma levels of factor VII, VIII, IX or
  XI
• Raised plasma levels of fibrinogen
• Raised plasma levels of homocysteine, possibly
  B12 defriciency
• Glucosylceramide deficiency
• Coagulation factor IX concentrates
• Lupus anticoagulant
• Oestrogen therapy (oral contraceptive and HRT)
• Heparin-induced thrombocytopenia
• Pregnancy and puerperium
• Surgery, especially abdominal and hip
• Major trauma
• Malignancy
• Myocardial infarct
• Thrombocythaemia

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Risk factors cont

• Related to stasis
• Cardiac failure
• Stroke
• Prolonged immobility
• Pelvic obstruction
• Nephrotic syndrome
• Dehydration
• Hyperviscosity, polycythaemia
• Varicose veins

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Risk factors cont

• Related to unknown factors
• Age
• Obesity
• unfitness
• Sepsis
• Paroxysmal nocturnal haemoglobinuria
• Behcets disease

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Risk factors

• Related to coagulation abnormality
• Hereditary hemostatic disorders
• Factor V Leiden
• Prothrombin G20210A variant
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
• Abnormal fibrinogen
• Abnormal plasminogen

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Factor V Leiden

• Activated protein C resistance
• Characterized by a poor anticoagulant response to
  activated protein C (APC)
• Specific G-to-A substitution at nucleotide 1691
  in gene for factor V. Predicts a single amino
  acid replacement (Arg506Gln) at one of three APC
  cleavage sites in the factor Va molecule
• Factor V Leiden is inactivated approx. ten times
  slower than normal factor V and persists longer
  in the circulation, resulting in increased
  thrombin generation and a mild hypercoagulable
  state
• Most common genetic defect for inherited
  thrombosis
• 15 of Causasians are carriers
• Rare in Asians Africans

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Factor V Leiden cont

• Diagnosis made either by the APC resistance assay
  as a coagulation screening test or by DNA
  analysis of the factor V gene
• Heterozygotes 5-10x ? risk of clotting
• Heterozygotes OCP 30 to 35 fold ? risk
• Homozygotes 50-100x ? risk
• Heterozygotes who are also heterozygous for the
  G20210A prothrombin mutation have eg 30x ? risk
• Homozygotes OCP ???gt100
• Hyperhomocysteinemia combined with Factor V
  Leiden, heterozygous 20 fold ? risk

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Risk factors

• Related to coagulation abnormality
• Hereditary hemostatic disorders
• Factor V Leiden
• Prothrombin G20210A variant
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
• Abnormal fibrinogen
• Abnormal plasminogen

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Protein C deficiency

• Protein C inactivates factor Va and factor VIIIa
• Two classifications of protein C deficiency type
  I and type II
• Type I protein C deficiency inadequate amount of
  protein C present
• Type II protein C deficiency normal amount but
  defective protein C molecules. Numerous defects
  in the protein C molecule have been described
  that alter its interactions with thrombomodulin,
  phospholipids, factor Va and factor VIIIa as well
  as others.

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Protein C deficiency

• Present in approximately 0.2 of the general
  population
• Heterozygous 7 fold incr. risk
• Homozygous serious thrombosis at birth (?FFP
  use, Protein C use)
• Both protein C and protein S are Vit. K dependent
• Diagnosis Protein C assays

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Risk factors

• Related to coagulation abnormality
• Hereditary hemostatic disorders
• Factor V Leiden
• Prothrombin G20210A variant
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
• Abnormal fibrinogen
• Abnormal plasminogen

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Protein S deficiency

• Function is carried out directly by protein C,
  and protein S serves as a cofactor. Activated
  protein C combines with protein S on the surface
  of a platelet.
• Protein S exists in two primary forms. One form
  is free and the other is bound to an additional
  protein. Only the free form of protein S is able
  to interact with protein C in the manner
  described above
• Three classifications of protein S deficiency
  type I, type II and type III
• Type I inadequate amount of protein S present in
  both free and bound forms. Protein S that is
  present functions normally.
• Type II characterized by defective protein S
  molecules. The amount of protein S present is
  normal, but it is unable to interact normally
  with the other molecules involved in coagulation
  to perform its function.
• Type III characterized by a low amount of free
  protein S, but an overall normal amount of total
  protein S.

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Protein S deficiency cont

• Incidence in general population is not known.
• In the Caucasian population, protein S deficiency
  has been found in between 1 to 5 of persons who
  have a venous thrombotic event
• Heterozygous 6 fold increase in risk
• Homozygous severe thrombosis at birth

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Risk factors

• Related to coagulation abnormality
• Hereditary hemostatic disorders
• Factor V Leiden
• Prothrombin G20210A variant
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
• Abnormal fibrinogen
• Abnormal plasminogen

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Antithrombin III deficiency

• Antithrombin is a potent inhibitor of the
  reactions of the coagulation cascade.
• Although the name, antithrombin, implies that it
  works only on thrombin, it actually serves to
  inhibit virtually all of the coagulation enzymes
  to at least some extent.
• The primary enzymes it inhibits are factor Xa,
  factor IXa and thrombin (factor IIa). Also has
  inhibitory actions on factor XIIa, factor XIa and
  the complex of factor VIIa and tissue factor.
• Its ability to limit coagulation through multiple
  interactions makes it one of the primary natural
  anticoagulant proteins.

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Antithrombin deficiency cont

• Antithrombin acts as a relatively inefficient
  inhibitor on its own.
• However, when it is able to bind with heparin,
  the speed with which the reaction that causes
  inhibition occurs is greatly accelerated
• Two primary types type I and type II.
• Type I characterized by an inadequate amount of
  normal antithrombin present.
• Type II normal amount of antithrombin present,
  but does not function properly and is thus unable
  to carry out its normal functions.
• In many cases, the antithrombin in type I
  deficiencies has a problem binding to heparin,
  although there have been multiple other changes
  to the antithrombin molecule described.
• Heterozygous 5 fold increase risk clots
• Homozygous type I thought to be lethal prior to
  birth
• Homozygous type II both arterial and venous
  thrombotic disease. Often have severe
  complications.
• ?Antithrombin concentrates

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Risk factors

• Related to coagulation abnormality
• Hereditary hemostatic disorders
• Factor V Leiden
• Prothrombin G20210A variant
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
• Abnormal fibrinogen
• Abnormal plasminogen

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Prothrombin (Factor II) G20210A variant

• Second most common genetic defect for inherited
  thrombosis
• Autosomal dominant disorder
• Heterozygotes 3- to 11-fold ? risk for thrombosis
  in both men and women and for all age groups.
  Homozygosity is rare
• Frequently co-inherited in Factor V Leiden
  carriers
• Nucleotide substitution of G to A at position
  20210 in the untranslated portion of the
  prothrombin gene on chromosome 11 causes an
  elevation of the level of functional prothrombin
  in plasma which is associated with an increased
  risk of thrombosis
• Diagnosed by a PCR test on WBCs

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Risk factor

• Hereditary or acquired haemostatic disorders
• Raised plasma levels of factor VII, VIII, IX or
  XI
• Raised plasma levels of fibrinogen
• Raised plasma levels of homocysteine
• Glucosylceramide deficiency
• Coagulation factor IX concentrates
• Lupus anticoagulant
• Oestrogen therapy (oral contraceptive and HRT)
• Heparin-induced thrombocytopenia
• Pregnancy and puerperium
• Surgery, especially abdominal and hip
• Major trauma
• Malignancy
• Myocardial infarct
• Thrombocythaemia

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Antiphospholipids

• LAs have traditionally been classified as
  anti-phospholipid antibodies, but a more correct
  view is that they are antibodies directed against
  plasma proteins, which also bind to phospholipid
  surfaces, a type of fat molecule that is part of
  the normal cell membrane.
• There are three test in which antiphospholipid
  antibodies may show up
• 1) anticardiolipin antibodies,
• 2) the lupus anticoagulant and
• 3) antibodies directed against specific molecules
  including a molecule known as beta-2-glycoprotein
  1. (also anti-prothrombin and the
  "false-positive" test for syphilis)

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Antiphospholipids cont

• They are usually IgG, IgM, or mixtures of both,
  and frequently interfere with standard
  phospholipid-dependent coagulation tests, often
  being discovered accidentally such as when a
  prolonged activated partial thromboplastin time
  (APTT) is found during a pre-operative
  evaluation. Importantly, the clotting test
  abnormalities caused by LA are in vitro
  phenomena the antiphospholipid antibodies of the
  LA react with the phospholipid preparations used
  to initiate clotting reactions. In vivo clotting
  factor activities are not diminished and, except
  in extremely rare cases where there are specific
  antibodies directed against clotting factor II,
  there is no danger of a bleeding diathesis.

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Antiphospholipids cont

• Often, there are no clinical consequences other
  than the need to explain the reason for the long
  APTT. A minority of patients with LA have a
  hypercoagulable state manifested by recurrent
  thromboses, multiple spontaneous miscarriages,
  migraine headaches, or stroke. Very rarely,
  patients may have bleeding. NB important cause of
  strokes in people under age 40.
• Two main classifications of antiphospholipid
  antibody syndrome If patient has an underlying
  autoimmune disorder (e.g. SLE), patient said to
  have secondary antiphospholipid antibody
  syndrome. If patient has no known underlying
  autoimmune disorder, it is termed primary
  antiphospholipid antibody syndrome

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Lupus Anticoagulant

• Suspect LA in patients with prolonged PTT and no
  history of bleeding, in women with recurrent
  miscarriages, and in those with recurrent DVT.
• LA not always associated with SLE, although about
  50 of SLE patients test positive for LA.
  However, many patients with LA do not have SLE or
  any other autoimmune disorder.
• The presence of LA does not necessarily indicate
  the diagnosis of antiphospholipid syndrome. The
  syndrome requires a positive test of an
  anti-phospholipid antibody (LA or anticardiolipin
  antibody), repeatably positive at least 6 weeks
  later (ie may be transient) plus a clinical
  history of pregnancy loss or thrombosisvenous
  more often than arterial.

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Investigations

• Screening of population is not justified because
  overall incidence of thrombosis is low
• Screen people in whom an occlusive event has
  occurred and has unusual circumstances e.g. young
  age, recurrent episodes, family hx

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When to test?

• At the thrombotic event or later?
• This is currently unresolved (debate exists)
• Acute thrombosis may give false positive
  clotting-based diagnosis
• Concurrent anticoagulation may complicate the
  picture

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Thrombophilia Laboratory Test Profile

• Order the following series of assays when the
  patient is not taking an anticoagulant such
  as warfarin or heparin and has taken no
  anticoagulant for at least ten days. Further, the
  following assays are valid only if the patient
  has not had a thrombotic event in the previous
  two weeks.
• Activated protein C resistance (APCR)go on to do
  the factor V Leiden mutation assay when the APCR
  ratio is below the lower limit of the reference
  interval, indicating resistance
• Antithrombin activityadd the antithrombin
  antigen assay when activity is consistently low
• Protein C activityadd the protein C antigen
  assay when activity is consistently low
• Protein S activityadd the free and total protein
  S antigen when activity is consistently low
• Factor VIII activity
• Lupus anticoagulant testing
• Plus the next slide shows tests that can be done
  whether or not the patient is on anticoagulants
  -gt

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Acute Thrombophilia Test Profile

• Can order the following assays when the patient
  is currently taking an anticoagulant such
  as warfarin or heparin or has taken an
  anticoagulant within the last ten days. Further,
  the following assays may be ordered if the
  patient has had a thrombotic event in the past
  two weeks
• factor V Leiden mutation DNA assay
• Prothrombin G20210A mutation
• Fasting homocysteine
• Anticardiolipin antibodies IgG and IgM
• May be able to do lupus anticoagulant assay while
  on warfarin (but not heparin)
• B12/folate