L09 Blood Haemostasis 4 Bleeding disorders Part 2

Overview

This lecture completes the bleeding disorders series. It covers the structure and function of von Willebrand factor, the classification, clinical features, laboratory diagnosis and treatment of von Willebrand disease, how platelets and the coagulation cascade are integrated into a single haemostatic response, Heparin as an injected anticoagulant working through antithrombin, and bleeding that arises from fragile vessels rather than from platelets or clotting factors. A set of worked mini-cases ties the three haemostasis lectures together.

von Willebrand factor: structure and synthesis

• The gene coding for vWF is on chromosome 12.
• vWF is a multimeric glycoprotein, MW 250 kDa.
• Synthesised in megakaryocytes and endothelial cells.
• Stored in the $\alpha$-granules of platelets and the Weibel-Palade bodies of endothelial cells respectively.
• The circulating form is a huge multimer containing up to 40 subunits.
• ADAMTS13 is a metalloprotease that cleaves the large vWF multimers.

Subunit domain structure, in order: D1, D2, D’, D3, A1, A2, A3, D4, B1-3, C1, C2, CK. Binding partners map to specific domains:

Domain	Binds
D’/D3	FVIII, heparin
A1	GPIb, heparin, collagen
A1-A2-A3 region	ADAMTS13 cleavage site
A3	collagen
C1/C2 (C-terminus)	GPIIb/IIIa

Disulphide (S-S) linkages join subunits at both ends of the multimer.

What vWF does

• vWF binds collagen, platelets and FVIII.
• vWF stabilises FVIII when bound to it. Unbound FVIII is easily degraded: half-life rises from about 2.4 hours when free to 12 hours when bound to vWF. Low vWF therefore produces low FVIII as a secondary effect.
• In a resting vessel, vWF multimers, free FVIII and resting platelets circulate; with injury, vWF adheres to the exposed subendothelial matrix.
• With shear forces of flowing blood, the vWF multimers uncoil and stretch along the matrix, platelets adhere in a chain via GPIb and become activated, clumping together.
• Reduced plasma vWF therefore causes reduced platelet adhesion, which causes reduced platelet aggregation, giving prolonged bleeding from cuts and increased bruising.

von Willebrand disease

Key points:

• Frequency 1:500 to 1:1000; about 1% of the population affected (the same figure is reported for NZ).
• Sex affects men and women equally.
• Inheritance inherited bleeding disorder, autosomal dominant or autosomal recessive depending on the type.
• Basis low levels of vWF function, either from a reduced level or absence of vWF protein (types 1 and 3) or from production of an abnormal protein (type 2). Severity of bleeding increases.

Clinical signs:

• Mucosal bleeding: frequent and prolonged nose bleeds (epistaxis); heavy and prolonged menstruation, with recurrent iron deficiency anaemia common in women.
• Easy bruising: increased size and frequency of bruises.
• Prolonged bleeding from cuts, wounds, dental treatment and surgery.

Laboratory testing in vWD

Screening tests: CBC, APTT, PT, fibrinogen.

Diagnostic tests:

• vWF:Ag measures the amount of vWF protein.
• vWF:Activity a functional assay that mimics binding to platelet.
• vWF:CB a function assay.
• FVIII:C a measure of the amount of FVIII in the blood.

vWD subtypes

Type	Defect	Inheritance	vWF:Ag	vWF:Activity	vWF:CB	FVIII:C
1	Partial quantitative deficiency of vWF	AD	mildly low to low	mildly low to low	mildly low to low	normal to low
2A	Point mutation giving a vWF variant with increased susceptibility to proteolysis by ADAMTS13	AD	normal to low	low to very low	low to very low	mildly low to low
2B	Mutations cause increased affinity of vWF for platelet glycoprotein	AD	normal to low	low to very low	low to very low	normal to low
2M	Qualitative variant with decreased platelet-dependent function, not caused by absence of vWF multimers	AD	normal to low	low to very low	low to very low	mildly low to low
2N	Qualitative variant with markedly decreased affinity for FVIII	AD	normal to low	normal to low	normal to low	low to very low
3	Complete deficiency of vWF	AR	absent	absent	absent	low

The pattern that separates the groups: type 1 lowers antigen and function together; type 2 lowers function out of proportion to antigen (except 2N, where the isolated casualty is FVIII); type 3 abolishes vWF entirely.

Haemophilia compared with vWD

	Haemophilia A or B	von Willebrand disease
Inheritance	X-linked	autosomal dominant
Defect	low FVIII or FIX	low vWF (mildly low FVIII)
Symptoms	joint and muscle bleeds	mucosal bleeding, cuts, trauma
PFA-100/200	normal	prolonged
APTT	high	normal or mildly prolonged

Treatment of vWD and mild platelet function defects

Two approaches:

1. Boost vWF/FVIII levels.
   • Desmopressin (DDAVP) stimulates endothelial release of vWF, giving a 2 to 5 fold increase, with temporary benefit lasting 2 to 8 hours.
   • Or a blood product, FVIII-vWF concentrate, if levels are very low.
2. Inhibit fibrinolysis with tranexamic acid. This is standard treatment for managing heavy menstrual bleeding, minor surgery and dental extractions.

Fibrinolysis and where tranexamic acid acts: blood vessel endothelium releases tPA; tPA converts plasminogen to plasmin; PAI (plasminogen activator inhibitor) inhibits tPA; plasmin acts on the fibrin clot, breaking it down into FDPs (fibrin degradation products). Tranexamic acid blocks the activation step generating plasmin, so the fibrin clot is preserved and a weak clot is supported.

Integration of platelets and coagulation

Haemostasis is a combination of platelets plus coagulation. Bleeding triggers three responses that converge on haemostasis being achieved and bleeding stopped:

1. Vessel constriction.
2. Platelets: activation and formation of the platelet plug.
3. Coagulation cascade activated, producing fibrin.

The end product is a platelet-fibrin clot: a fibrin mesh studded with entrapped platelets.

Warning

One slide in this sequence carries no readable title or caption; it groups a blood smear showing platelets, a photo of platelet-rich blood product bags, and an electron micrograph of a fibrin mesh with platelets. The pairing (platelets plus blood product leading to a platelet-fibrin clot) is inferred from the images, not stated.

Platelets supply the surface for coagulation

Coagulation factors need phospholipid membranes for their activity, mostly supplied by activated platelets. Increased phosphatidylserine exposure creates a negatively charged surface for coagulation. In the cascade, phospholipid is required at the IX/VIIIa to X step and at the prothrombin to thrombin step. The cascade as drawn: XII to XI; the extrinsic limb (VII) and the intrinsic limb (XI, IX) converge on X; X with Va gives Xa; prothrombin with phospholipid gives thrombin; fibrinogen gives fibrin. VIIIa and Va are the activated cofactors.

Warning

The platelet diagram accompanying this cascade is small and partly cropped; only the labels “4 $\alpha$IIb$\beta$3”, “Thromboxane-A2”, “Arachidonic acid” and numbered markers 2 and 5 are legible.

Platelet granule contents

1. Serotonin for vasoconstriction and activation of adjacent platelets.
2. ADP to activate adjacent platelets.
3. Von Willebrand factor.
4. Fibrinogen and factor V to enhance coagulation. These feed directly into the cascade at the Xa/Va step and at the fibrinogen to fibrin step.
5. P-selectin for adhesion.
6. Platelet-derived growth factor to recruit fibroblasts for healing.

Platelet receptors and the thrombin burst

Thrombin generated by the coagulation pathway activates platelets via thrombin receptors, the “thrombin burst” produced by Xa/Va acting on prothrombin. Platelet receptors:

1. vWF initial adhesion and activation.
2. Collagen initial adhesion and activation.
3. ADP secreted by adjacent platelets.
4. Thrombin a product of coagulation.
5. Thromboxane (TXA2) secreted by adjacent platelets.
6. Fibrinogen formation of bridges between adjacent platelets.

The accompanying platelet membrane diagram shows multiple receptor types embedded in the membrane with intracellular signalling and activation arrows. The labels legible on it are: thromboxane A2, TP$\alpha$-R, PAR-4, P2Y1, P2Y12, ADP, and TXS and COX-1 inside the platelet. The slide does not state which of these receptors binds which ligand.

Warning

The receptor diagram labels are very small and partly illegible at the rendered resolution beyond the terms listed.

This is a positive feedback loop: coagulation makes thrombin, thrombin activates platelets, and activated platelets provide the phospholipid surface and the granule fibrinogen and factor V that drive further coagulation.

Worked case: 10-year-old boy with easy bruising

Referred to paediatrics for easy bruising, with multiple bruises over knees, shins and arms, photographs of the bruises provided by his mother. His mother also bruises easily but has no formal diagnosis. There is no risk of non-accidental injury.

Tests chosen: platelet count, coagulation screen (PT, APTT, fibrinogen), then PFA-100/200 and vWF levels.

Results:

• Platelets 327 and 225 (ref 150 to 425 x10
• PT 10.3 s both occasions (ref 9.0 to 13.0); INR 1.0 (0.8 to 1.2); APTT 27 s (ref 20 to 31); fibrinogen 4.2 then 2.4 g/L (ref 1.7 to 4.3). All normal.
• PFA closure times: collagen/adrenaline >298 s (ref 82 to 160); collagen/ADP >294 s (ref 62 to 120). Both markedly prolonged.
• Factor VIII:C 122 and 133 IU/dL (ref 50 to 150), normal.
• vWF antigen 80% and 83% (ref 50 to 200), normal.
• vWF activity 31% and 36% (ref 50 to 200), low.

Diagnosis: type 2 vWD. Normal antigen with low activity is the discriminating pattern.

Heparin

Key points:

• A polysaccharide (a sulphated polysaccharide), specifically built of repeating sugar units: D-glucosamine, D-glucuronic acid, L-iduronic acid and N-acetyl-D-glucosamine with sulphate and carboxyl substituents.
• Short half-life of approximately 50 minutes, and it must be injected. It has immediate anticoagulant effect.
• Binds to antithrombin and increases antithrombin activity by about 300-fold.
• Through antithrombin it inhibits FIIa (thrombin), FXa, FIXa and FXIa. Factors VIIIa and Va are not inhibited.
• 1 unit/mL heparin will block coagulation completely by this mechanism.

Structural requirement differs by target: unfractionated heparin contains a pentasaccharide sequence which binds antithrombin. Inhibition of factor Xa requires only that pentasaccharide sequence, whereas inhibition of thrombin requires the full heparin chain length.

Monitoring: heparin dose control is needed to achieve the appropriate level of anticoagulation. APTT provides a useful clinical measure of heparin effect, with linear prolongation of APTT as dose increases across the range 0 to 0.3 U/mL.

Vascular bleeding

Not all bleeding is related to platelets and coagulation. Bleeding can arise from fragile blood vessels:

• Senile purpura bleeding from small blood vessels that are poorly supported by connective tissue.
• Prolonged steroid medication thinning of connective tissues.
• Scurvy defective collagen production.
• Hereditary haemorrhagic telangiectasia small vascular malformations in the skin and in the mucosal linings of the nose and gastrointestinal tract.
• Angiodysplasia vascular malformations in the gut, often causing iron deficiency; common in the elderly.
• Vasculitis (inflammation of the vessel wall) vascular damage from immune complexes (IgG plus antigen plus complement) in the basement membrane. Example: IgA vasculitis, often post-viral in childhood. Histology shows an inflamed vessel with surrounding inflammatory cell infiltrate; clinically the legs show multiple small red or purple spots (petechiae and purpura).

Mini-cases with answers

These are provided to test knowledge in your own time, using material from all three haemostasis lectures to identify relevant information and reach a diagnosis or differential diagnosis.

Case 1. 22-year-old woman, persisting nose bleed (4 h) and a rash of red spots 2 to 4 mm diameter that do not blanch with pressure, appearing over the past 48 h. Blood screen normal apart from the platelet count. Platelets 6 (150 to 400 x10^9/L); PT 9.0 (9.0 to 13.0 s); APTT 27 (22 to 34 s); fibrinogen 2.7 (1.8 to 4.0 g/L). Severe thrombocytopenia. Most likely possibilities: autoimmune or drug-induced thrombocytopenia; malignant disease of marrow (leukaemia or lymphoma), though these are not likely if the blood count is otherwise normal.

Case 2. 52-year-old man, generally unwell with increased bruising. Hb 120 (135 to 175 g/L); platelets 150; PT 24 (9.0 to 13.0 s); APTT 45 (22 to 34 s); APTT on 50% patient plus 50% normal plasma 33 s; fibrinogen 2.0. Changes: both the intrinsic and extrinsic pathways have prolonged clotting, and correction of the APTT with normal plasma indicates a deficiency of one or more factors. Most likely: acute or chronic liver disease depressing multiple factors; also consistent with malabsorption or negligible food intake for more than 2 weeks (vitamin K deficiency).

Case 3. 22-year-old man with a large thigh muscle haematoma after a rugby game and a history of large bruises after trauma. PT 12; APTT 46 (22 to 34 s); fibrinogen 2.2; platelets 193; vWF all within reference intervals; PFA-100/200 normal closure times. Most likely: a defect in the intrinsic coagulation pathway, probably a haemophilia. Specialist factor assays: factor VIII 20% (50 to 150%), factor IX 85% (normal). Diagnosis: mild haemophilia A with traumatic muscle bleeding; he needs to give up contact sports.

Case 4. 32-year-old woman who has always bruised readily, has heavy periods and has been treated repeatedly for iron deficiency. PT 10.1; APTT 37 (22 to 34 s); fibrinogen 3.2; platelets 325; Hb 105 (115 to 155 g/L); PFA-100/200 prolonged closure times >300 s. Summary: impaired platelet function with a mild defect of the intrinsic pathway. Likely: von Willebrand disease or a platelet function defect. Example specialist findings: factor VIII 35% (50 to 150%), vWF antigen 22%, vWF activity 18%. Both vWF protein and function are reduced to a similar degree, giving vWD type 1; FVIII is mildly low because it is bound to vWF and decays faster when vWF levels are low.

Case 5. Patient with renal failure on haemodialysis, given a heparin infusion during the procedure to anticoagulate and stop clotting on the dialyser membrane and in the tubing. Pre then during: Hb 85 then 88 (115 to 165 g/L); platelets 185 then 176; PT 10.5 then 11.0; APTT 24 then 85 (22 to 34 s); fibrinogen 2.8 then 2.7. Changes: moderate anaemia, secondary to renal failure and lack of erythropoietin which is mostly produced by the kidneys; APTT prolonged during dialysis to about twice normal due to heparin, as intended. Heparin is an injectable sulphated polysaccharide anticoagulant with immediate effect. It acts by binding antithrombin and enhancing its effect about 300-fold. Half-life is about 50 minutes, so heparin will disappear over 3 to 5 hours after the infusion stops.

Case 6. 8-year-old girl, previously well with no unexpected health problems, now with numerous bruises and petechiae. Blood count: Hb 125 and neutrophils 4.3 x10^9/L, both normal. Platelets 5 (150 to 400 x10^9/L); fibrinogen 2.0 (1.8 to 4.0 g/L); APTT 30 (22 to 36 s); PT 10.5 (9 to 13 s). Differential: isolated thrombocytopenia, and the history suggests it is acquired, so almost certainly immune thrombocytopenia; rarely acute viral infection or medication could cause isolated thrombocytopenia. Further tests: no, unless the child failed to improve or respond to treatment.

Important

The screening panel does most of the diagnostic work. Isolated low platelets points to thrombocytopenia; isolated prolonged APTT with normal PFA points to haemophilia; prolonged PFA with a mildly prolonged APTT points to vWD or a platelet function defect; prolonged PT and APTT that correct with normal plasma points to multiple factor deficiency from liver disease or vitamin K deficiency.

Self-test

1. State where vWF is synthesised and where it is stored in each cell type.
2. Explain why low vWF levels produce a low FVIII level, giving the relevant half-life figures.
3. Describe what happens to vWF and platelets at a site of vessel injury under shear, in order.
4. Name the vWF domains that bind FVIII, GPIb and collagen, and state which enzyme cleaves vWF multimers.
5. State the frequency of vWD, its sex distribution and its inheritance patterns.
6. List the clinical signs of vWD by category.
7. Distinguish the underlying defect in type 1, type 2 and type 3 vWD.
8. Given a patient with normal vWF:Ag but vWF:Activity of 31% and normal FVIII:C, which vWD type is this and why?
9. Distinguish vWD type 2N from the other type 2 variants by its laboratory pattern.
10. Distinguish haemophilia A from vWD on inheritance, symptoms, PFA-100/200 and APTT.
11. Name the four diagnostic tests used in vWD and state what each measures.
12. Describe the two treatment approaches for vWD, naming the drugs and the magnitude and duration of the desmopressin effect.
13. Describe the fibrinolytic pathway in order and explain where tranexamic acid acts.
14. List the three responses to bleeding that converge to achieve haemostasis.
15. Explain what activated platelets contribute to the coagulation cascade at the membrane level, and name the two cascade steps that require phospholipid.
16. List the six platelet granule contents with the function of each.
17. List the six platelet receptors covered and state what activates each.
18. What is heparin chemically, how is it given, and what is its half-life?
19. Explain heparin’s mechanism of action, including which factors are inhibited and the fold enhancement.
20. Explain why inhibiting thrombin requires a longer heparin chain than inhibiting factor Xa.
21. How is heparin therapy monitored, and what is the relationship between dose and that test?
22. List the causes of vascular bleeding covered, with the vessel or connective tissue defect in each.
23. A 52-year-old man has PT 24 s and APTT 45 s, and the APTT corrects to 33 s on mixing with normal plasma. What does the correction tell you, and what are the likely diagnoses?
24. A 22-year-old man has an isolated prolonged APTT of 46 s with a normal PFA closure time and normal vWF. What is the likely diagnosis and what test confirms it?
25. Integrative: a 32-year-old woman has prolonged PFA closure times, a mildly prolonged APTT, iron deficiency anaemia and heavy periods. Explain how her platelet-side and coagulation-side abnormalities arise from a single underlying defect.

Answers

Reveal answers

1. vWF is synthesised in megakaryocytes and endothelial cells. It is stored in the $\alpha$-granules of platelets and in the Weibel-Palade bodies of endothelial cells respectively.
2. vWF stabilises FVIII when bound to it, and FVIII is easily degraded when unbound. FVIII half-life is about 2.4 hours when free and 12 hours when bound to vWF, so low vWF gives low FVIII as a secondary effect.
3. With injury, vWF adheres to the exposed subendothelial matrix. With shear, the vWF multimers uncoil and stretch along the matrix. Platelets then adhere via GPIb, forming a chain, become activated and clump together.
4. FVIII binds at D’/D3; GPIb binds at A1; collagen binds at A1 and A3. ADAMTS13 is the metalloprotease that cleaves the large multimers, at a site in the A1/A2/A3 region.
5. Frequency 1:500 to 1:1000, about 1% of the population (same reported for NZ). It affects men and women equally. Inheritance is autosomal dominant or autosomal recessive depending on the type (types 1 and 2 AD, type 3 AR).
6. Mucosal bleeding (frequent and prolonged epistaxis; heavy and prolonged menstruation, with recurrent iron deficiency anaemia common in women); easy bruising (increased size and frequency of bruises); prolonged bleeding from cuts, wounds, dental treatment and surgery.
7. Type 1 is a partial quantitative deficiency of vWF. Type 2 is production of an abnormal protein, a qualitative defect. Type 3 is complete deficiency of vWF.
8. Type 2 vWD. The antigen level shows the protein is present in normal amount but the activity assay shows the protein does not function, which is the qualitative pattern of type 2 rather than the parallel reduction of type 1.
9. Type 2N is a qualitative variant with markedly decreased affinity for FVIII, so vWF:Ag, vWF:Activity and vWF:CB are normal to low while FVIII:C is low to very low. The other type 2 variants have low to very low activity and CB with a comparatively better preserved FVIII:C.
10. Haemophilia is X-linked, vWD autosomal dominant. Haemophilia causes joint and muscle bleeds; vWD causes mucosal bleeding, and bleeding from cuts and trauma. PFA-100/200 is normal in haemophilia and prolonged in vWD. APTT is high in haemophilia and normal or only mildly prolonged in vWD.
11. vWF:Ag measures the amount of vWF protein; vWF:Activity is a function assay mimicking binding to platelet; vWF:CB is a function assay; FVIII:C measures the amount of FVIII in the blood.
12. First, boost vWF/FVIII levels: desmopressin (DDAVP) stimulates endothelial release of vWF giving a 2 to 5 fold increase with temporary benefit for 2 to 8 hours, or a FVIII-vWF blood product if levels are very low. Second, inhibit fibrinolysis with tranexamic acid, standard for heavy menstrual bleeding, minor surgery and dental extractions.
13. Endothelium releases tPA; tPA converts plasminogen to plasmin; PAI inhibits tPA; plasmin degrades the fibrin clot into fibrin degradation products. Tranexamic acid blocks the activation of plasmin, so the fibrin clot is preserved and a weak clot is supported.
14. Vessel constriction; platelet activation and platelet plug formation; activation of the coagulation cascade producing fibrin. All three converge on bleeding stopped and haemostasis achieved.
15. Activated platelets supply the phospholipid membrane surface that coagulation factors require, through increased phosphatidylserine exposure creating a negatively charged surface. Phospholipid is required at the IX/VIIIa to X step and at the prothrombin to thrombin step.
16. Serotonin for vasoconstriction and activation of adjacent platelets; ADP to activate adjacent platelets; von Willebrand factor; fibrinogen and factor V to enhance coagulation; P-selectin for adhesion; platelet-derived growth factor to recruit fibroblasts for healing.
17. vWF for initial adhesion and activation; collagen for initial adhesion and activation; ADP secreted by adjacent platelets; thrombin, a product of coagulation; thromboxane TXA2 secreted by adjacent platelets; fibrinogen, forming bridges between adjacent platelets.
18. Heparin is a sulphated polysaccharide. It must be injected and has immediate effect. Half-life is approximately 50 minutes, so it disappears over 3 to 5 hours after an infusion stops.
19. Heparin binds to antithrombin and increases antithrombin activity by about 300-fold. The enhanced antithrombin inhibits FIIa (thrombin), FXa, FIXa and FXIa. At 1 unit/mL heparin blocks coagulation completely.
20. Antithrombin binding requires only the pentasaccharide sequence, and that alone suffices for the complex to inhibit factor Xa. Inhibiting thrombin requires the full heparin chain length rather than the pentasaccharide alone.
21. Heparin is monitored with the APTT, which provides a useful clinical measure of heparin effect. APTT prolongs roughly linearly with increasing heparin concentration over 0 to 0.3 U/mL, so dose can be titrated against it.
22. Senile purpura, from small vessels poorly supported by connective tissue; prolonged steroid medication, thinning connective tissues; scurvy, defective collagen production; hereditary haemorrhagic telangiectasia, small vascular malformations in skin and in nasal and gastrointestinal mucosa; angiodysplasia, vascular malformations in the gut often causing iron deficiency, common in the elderly; vasculitis, vascular damage from immune complexes (IgG plus antigen plus complement) in the basement membrane, for example post-viral IgA vasculitis in childhood.
23. Both intrinsic and extrinsic pathways show prolonged clotting. Correction of the APTT with normal plasma indicates a deficiency of one or more factors. Likely diagnoses are acute or chronic liver disease depressing multiple factors, or vitamin K deficiency from malabsorption or negligible food intake for more than 2 weeks.
24. A defect in the intrinsic coagulation pathway, probably a haemophilia. Factor assays confirm it: in the case given, factor VIII 20% (50 to 150%) with normal factor IX at 85%, diagnosing mild haemophilia A. He needs to give up contact sports.
25. She has von Willebrand disease, type 1 in the worked example (vWF antigen 22% and activity 18%, reduced to a similar degree). Low vWF reduces platelet adhesion to the vessel wall, prolonging the PFA closure time, which is the platelet-side abnormality. The same low vWF fails to stabilise FVIII, which then decays faster, giving a mildly low FVIII of 35% and the mildly prolonged APTT on the coagulation side. The chronic mucosal bleeding, here heavy periods, produces the iron deficiency anaemia.