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1. Prevention of blood loss from intact vessels
   
   • Through the relationship between sound vessel walls and platelets
2. Arrest of bleeding from damaged vessels
   
   • Blood vessel reaction to injury
   • Platelet plug formation at the site of damage
   • Plasma proteins causing coagulation

Components Of The Normal, Healthy Haemostatic Mechanism

1. Vascular endothelium (vasoconstriction)
2. Platelets (adhering and aggregating)
3. Coagulation Factors (in the plasma which lead to coagulation)
4. Fibrinolysis
5. Inhibitors

• Injury to the vascular wall leads to a local contractile response from smooth muscle cells induced by noradrenaline and serotonin. This mechanism is not sufficient to prevent blood loss, because vessels consists of capillaries which do not contain SMCs. Injury also leads to platelet released thromboxane A₂ (a vasoconstrictor)
• Later, prostacyclin is released which counters the effects of thromboxane A₂

• Platelets adhere to the exposed basement membrane and connective tissue and change from their dicoid shape.
• Secretion of ADP, serotonin, phospholipid and synthesis of platelet factors then occurs.
• Phospholipase C is activated, Ca²⁺ is liberated and phospholipase A2 is then activated liberating arachidonic acid which is essential to the production of thromboxane A₂ (TXA2)
• TXA2 and serotonin support the formation of reversible Plt aggregates and together with plasma clotting factors (eg VIII) irreversible plugs are formed.

• Fibrin is eventually completely digested by enzymes from the plasma and from cells.
• Endothelium replaces the fibrin

When investigating haemostatic disorders it is important to determine the following points about the patients history:

• Age at which symptoms began (rapid onset post-partum usually indicates inheritance of disease)
• Persistence and severity of symptoms
• Family history
• Exclude associated disease eg. aplastic anaemia, leukaemia, malignancy, uraemia, liver disease or infections
• History of drug therapy
• Past or recent exposure to toxic chemicals eg. benzene, insect sprays, hair dyes etc

Coagulation Factor Disorders

• These factors are plasma proteins which may be absent, present in only reduced amounts or dysfunctional

1. Hereditary - from a gene on the X chromosome
2. Autosomal - from a gene on a chromosome other than X or Y
3. Acquired - These are among the more common defects, and bleeding usually occurs from more than one site.

Hereditary Disorders

• Usually inherited as only a single factor defect/deficiency, resulting in bleeding from only one site
• Severe clinical symptoms usually different. Tends to manifest as bleeding in deep tissues (eg. haemarthroses, intracranial bleeding, GIT haemorrhage, haematoma or delayed bleeding) from arteriole rupture rather than the more superficial bleeding with eg platelet disorders.
• Clinical symptom strength usually proportional to the level of the deficiency

• The circulating molecule consists of 2 non-covalently linked sub-units which are inherited differently (ie from different chromosomes)
• Factor VIII molecule is also known as the "procoagulant cofactor" (VIII:C - old nomenclature) or the Factor VIII / von Willebrand's (vWF) factor complex (see Figure).

General Information
	von Willebrand's Factor	Factor VIII
Inheritance Pattern	Chromosome 12 Autosomal Dominant (usually)	Sex-linked recessive (X-linked) Lyon hypothesis protects females
Synthesis	In endothelial cells and megakaryocyte cytoplasm	Liver (along with nearly all other factors)-See figure above
Properties	(i) Important in binding platelets to exposed collagen via "Glycoprotein Ib" (ii) Acts as a carrier for VIII (stabilising it and preventing its decay)	Cofactor in the intrinsic p'way
Molecular Weight	Very large - has high and low molecular weight subunits-multimeric(see figure above)	Comaparatively small
Antigen	vWF : Ag	F VIII : Ag
Lab Aspect	Causes agglutination of platelets with Ristocetin	aPTT detects VIII levels

Deficiency States
	vW Disease (vWD)	Haemophilia A
Clinical Manifestations	Symptoms vary within the propositus (person under study) and between siblings/kindred Epistaxis   Gingival bleeding Menorrhagia (xs uterine bleeding)   Easy bruising In sever cases: Haemarthroses   Spontaneous deep bleeding	Severity proportional to deficiency <1% normal VIII = Severe ..Haemarthroses (+ intense pain) ..Haematoma ..Cranial haemorrhages (25% die) -seen soon after birth 2-5% = Moderate (trauma induced) 6-25% = Mild (trauma induced)
Level of Deficiency	Usually 15-60%
Types of Deficiency	Type I Most common approx 70% of those seen decreased production of whole molecule Type IIa-f Loss of high MW units Type III Very rare ?Association with inter-family marriage Absence of vWF (some have abnormal molecules) Symptoms of a haemophilia Less than 2% vWF	Incidence of 1:5000 males - not always traceable via inheritance patterns 25% via mutation of genes Normal vWF - bleeding due to delayed fibrin formation
Laboratory Tests	Platelet Count - N BT - Inc (variable) PT - N aPTT - Inc (variable) Plt Aggregation Studies - Collagen: N; Collagen: N; Adrenalin: N; Ristocetin: AN Ristocetin Cofactor Assay: AN (Dec) vWF:Ag-Dec F VIII Assay: Dec	Plt Ct: N BT: N (may be delayed) PT: N; aPTT: Inc; Plt Aggregation Studies - Ristocetin: N Ristocetin Cofactor Assay:N vWF:Ag-N F VIII Assay: AN
Treatment	Recominant DNA-produced factor VIII
	Cryoprecipitate-Plasma from single donors, frozen and then slowly thawed. Last of the ice crystals (ppt) contain vWF, VIII and I. These crystals are made up to 20mL with donor plasma and infused back to the donor	F VIII concentrate -Commercially produced from pooled donors (expensive, short half-life, Ab to foreign proteins and risk of disease)
	DDAVP (deamino-D-arginine vasopressin) - synthetic antidiuretic hormone which stimulates endothelial cells to release their stored vWF. Endothelial cells can become exhausted as the cell cannot maintain the levels required	Cryoprecipitate-8-10 hour half-life. At least 25% of recipients develop Ab to the concentrate as it is considered foreign-may need an immune suppressant
	Gene Therapy-In vitro experiments show promise for animal model and perhaps for clinical trials

Autosomal Disorders

• Known as "Factor I"
• Fibrinogen is present in the blood stream in the greatest quantity of any of the clotting factors (1-4g/L) and is considered to be the anchor of the clotting pathway.

Disorders Associated with Fibrinogen

• No detectable fibrinogen (less than 0.1g/L)
  
• Autosomal recessive
  
• Usually manifests itself at birth showing umbilical and mucous membrane bleeding
  
• Severity decreases with age

• 0.2-1g/L
  
• Autosomal recessive
  
• No treatment required unless injury occurs

• Abnormal function but normal levels
  
• Autosomal dominant
  
• Only problematic in cases of trauma-induced bleeding

Test	1	2	3
BT	P	N	N
PT	P	P	P
aPTT	P	P	P
TCT	P	P	P
Functional Fibrinogen	0.1g/L	0.2-1g/L	0.1g/L
Total Fibrinogen	Absent	0.2-1g/L	N
Plt Aggregation	AN	N	N

P=Prolonged; AN=Abnormal; N=Normal; BT=Bleeding Time; PT=Prothrombin Time; aPTT=Activated Partial Thromboplastin Time TCT=Thrombin Clotting Time   Thrombin is added to citrated patients plasma and the time for a clot to form is measured. A prolonged time indicates a deficiency in Factor I, dysfibrinogenaemia or the presence of inhibitors.

Platelet Aggregation:

Ristocetin (Uses Ristocetin and patient's PRP) - Requires vWF for aggregation but this test does not allow for AN patient platelets
Ristocetin Cofactor Assay (Uses patient PPP, Ristocetin and N PRP - to provide platelets) - Looks for vWF in patients plasma, discounting AN platelet function / structure

• Autosomal recessive
  
• Disease is detected 24-48 hours post-partum in the most severe cases. Bleeding will be seen from the umbilicus and in severe cranial haemorrhaging.
  
• Factor XIII's involvement with a clot often takes 24-48 hours. As none of the previous tests will tells us about this factor, a specific test is used.
  
• Clotting in plasma (PPP) is activated by the addition of calcium. The clot is then left to sit in a 5M solution of urea overnight. If the clot has dissolved, the patient has a Factor XIII deficiency. If the clot remains, Factor is present and functioning.

• Fresh frozen plasma (FFP). Factor XIII has a half-life of 3-7 days.

• Also known as "Haemophilia B"
  
• Factor IX deficiency is an intrinsic defect. Patients still have a normal primary haemostatic mechanism, but activation of fibrin clotting is delayed or inadequate.
• The disorder is linked to the X-chromosome.

Test	Factor IX Deficiency
BT	N to P
PT	N
aPTT	P
Plt.	N
Mix	N
IX	Decreased

P=Prolonged; AN=Abnormal; N=Normal; BT=Bleeding Time; PT=Prothrombin Time; aPTT=Activated Partial Thromboplastin Time; TCT=Thrombin Clotting Time; Plt=Platelet Count; IX=Factor IX Assay Mix=Mixing Sudies Uses patients plasma and mixes with normal plasma (1:1). Repeat aPTT - if the time has returned to normal, the problem has been corrected, telling us that we have a factor deficiency, not an inhibitor in the patient's plasma.

Treatment:

Given fresh plasma concentrate to provide a functional fibrin precursor.

• Usually involve more than one factor

Liver Disease

• Decreased synthesis of normal coagulation factors (except von Willebrands factor)
• Decreased levels of plasminogen
• Decreased levels of plasminogen activators
• Increased synthesis of fibrinogen but dysfibrinogenaemia
• Decreased clearance of activated factors
• Thrombocytopaenia

Lab Investigations:

Test	Liver Disease
BT	P
PT	P
aPTT	P
TCT	P
Plt.	Decreased
FDP	Increased

• Need fat to help absorption as well as bile
• Vitamin K1 - From vegetables
• Vitamin K2 - From bacteria in the gut

Causes:

• Malabsorption of fat
• Malnutrition
  • In the elderly
  • In patients receiving some oral antibiotics
• Newborns ((i)fat does not travel well in mother's milk (ii) almost sterile gut)
• Anticoagulants using vitamin K antagonists

Background:

• Vitamin K dependent plasma proteins - II, VII, IX, X, protein C and protein S
  • Appears important in the conversion from zygomatic form which requires gamma carboxylation
  • Protein can still be measured/produced but is not functional
  • Oral anticoagulants can interfere with vitamin K recycling via an antagonistic effect
  • Once the half life expires, factors start disappearing. First VII then IX & X then II
• Protein C & S
  • Protein S acts as a cofactor for the interaction between Thrombin and Thrombomodulin. The product of this reaction activates Protein C which destroys V and VIII, shutting down Thrombin production.
  • A deficiency of one or both results in thrombosis as there is nothing to stop clot formation. Oral anticoagulants will stop protein C/S production, thus they may increase thrombosis (may lead to ulceration due to skin necrosis)
  • Takes 5-6 hours to stop the extrinsic p'way (VIII), 2-40 to stop the intrinsic p'way (IX)

Disseminated Intravascular Coagulation (DIC)

• Damage to tissues or organs releases thromboplastin which triggers coagulation - mainly affecting the microcirculation
• DIC causes bleeding (due to massive reactive fibrinolysis)
• Tissue Damage releases Tissue Plasminogen Activator producing Plasmin from Plasminogen.
• Results in large amounts of degradation products (esp D-dimer) from fibrinolysis (also acting on V, VIII and I)
• breakdown interferes with further coagulation due to factor degradation

Causes:

• Infections and sepsis
• Pregnancy complications - toxaemia, amniotic fluid embolism
• Neoplasms - especially malignant eg. leukaemia
• Massive tissue injury - burns, extensive surgery
• Vascular injury
• Miscellaneous - snake bites, heat stroke

Diagnosis:

Test	DIC
PT	Inc
D-dimer	Inc
Schistocytes & Microspherocytes	Present
aPTT	Inc
Plt.	Dec
Fibrinogen Assay	Dec

Treatment:

• FFP, platelets etc are used to support lost elements

HELLP Syndrome:

• Haemolysis Elevated Liver Enzymes, Low Platelets
• A form of acute DIC sometimes in latter stages (must be born quickly) or just after, the birth of a baby

Inhibitors

• Naturally occurring

1. Anti-Coagulation
   1. Anti Thrombin (AT)
      • Most common inhibitor deficiency
      • Autosomal dominant inheritance of the deficiency
      • Deficiency leads to thrombosis (arterial or venous) - homozygous seen at birth
      • Purpura fulminans - widespread thrombotic episode
      • Heterozygous deficiency seen at a rate of 1:2000 (where levels fall to 40-60% of normal)
        -Tendancy to develop deep venous thrombosis (calf pain and ankle oedema). Aggrevated by stasis
      • Pregnancy and oral contraceptives decrease anti thrombin III
        Treatment:
      • Oral anticoagulant (often for life). May need hospitalisation and heparin if severe
   2. Protein C and Protein S show a similar pattern in deficiency (heterozygous deficiency more common)
   3. Lupus Anticoagulant (LAC)
      • Acquired antibody
      • Found in:
        <10% of SLE patients
        The elderly
        Pregnant women
        Autoimmune disease
        Lymphoproliferative disease
        Spontaneous in some healthy people
        
      • Commonly found accidentally (may often have no clinical symptoms-usually found in lab.
      • Patient may undergo surgery with no problems
      • The LAC antibody acts against phospholipid and the associated calcium dependent proteins
      • In the aPTT, the LAC antibody combines with the phospholipid surfaces of test reagents causing prolonging of the clotting time
        Diagnosis:
        • aPTT - Prolonged
        • Mixing Studies - not corrected (indicating inhibitor problems rather than a deficiency)
        • A small number of people with this develop thrombosis (in vivo acts against platelets and endothelial cell walls causing aggregation)
2. Anti-Fibrinolysis
   1. Anti-Plasmin