Esoteric Coagulation Testing Alphabet Soup: HIT, TTP, and VWD

1
Esoteric Coagulation TestingAlphabet Soup HIT,
TTP, and VWD

• Ken Friedman, MD
• Director, Hemostasis Reference Lab, Blood Center
  of Wisconsin

2
Educational Questions

• What is role of lab testing in evaluation and
  management of HIT?
• Is ADAMTS13 testing of value in TTP?
• When are supplemental tests called for in the
  evaluation of VWD?

3
Case 1

• 46 y o female with stage IB Endometrial CA
• TAH BSO and node dissection performed
• Given post-op UF heparin prophylaxis x 4 day
• Post-op day 6 has chest pain/dyspnea
• Helical CT reveals pulmonary embolism and DVT
• Platelet count is 239,000
• IV heparin reinitiated
• Day 9 patient develops leg pain with edema
• Platelet count is 39,000
• Hematology consult called

4
Questions to consider

• What is the role of laboratory testing?
• A. In establishing a diagnosis
• B. In management of anticoagulation therapy

5
Heparin-Induced ThrombocytopeniaClinical
Features

• Immune disease
• Neo-epitope (HeparinPF4) causes transient
  autoimmune disease
• Takes days to develop 4-14 days into heparin
  therapy
• May occur faster if recent heparin exposure
• May occur after heparin stopped (Delayed-onset
  HIT)
• Occurs 1-3 of patients on UFH, rarer with LMWH
• Moderate thrombocytopenia (rarely lt 20K)
• Suspect if platelet count falls by half from
  baseline (in medical patient) or from post-op
  peak (surgical patient)
• Associated with thrombotic events
• Increases risk for thrombosis 10-40 fold

6
Pathophysiology
Platelet
alpha granule
PF-4/heparin complex
IgG
PF-4 binds to surface of platelet following
activation
Complexes of heparin (GAG) and PF-4 molecules form
IgG binds to the PF-4/ heparin complex
Fc receptor
microparticles
IgG/PF-4/heparin complex activates via the Fc
receptor
Fc stimulation leads to the generation of
procoagulant-rich microparticles
Courtesy of Dr John G. Kelton, McMaster
University.
Hirsh et al. Arch Intern Med. 2004164361-369.
7
Monitoring for HIT
Heparin rx
ACCP 2004 Platelet counts should be monitored
during heparin therapy
8
How can you differentiate HIT from other causes
of thrombocytopenia?

• The problem
• Thrombocytopenia occurs in 15-40 of ICU patients
• Most receiving some form of heparin
• Keys to differentiating thrombocytopenia
• Is timing of thrombocytopenia consistent with
  HIT?
• Is degree of thrombocytopenia mild or severe?
• Is an alternative diagnosis more likely?
• Underlying illness (sepsis, infection,DIC, etc)
• Dilutional (from resuscitation or post-CABG)
• Did a new thrombosis occur on anticoagulation?
• Thrombosis occurs in half of HIT patients

9
Clinical presentations of HIT

• Isolated Thrombocytopenia
• Thrombosis
• Deep venous thrombosis (50)
• Pulmonary embolism (25)
• Acute thrombotic stroke or myocardial infarction
  (3-5)
• Acute limb ischemia (5-10)
• Adrenal hemorrhage/insufficiency (3-5)
• Other HIT syndromes
• Skin lesions at injection site (10-20)
• Warfarin-associated venous limb gangrene (5-10)
• Acute systemic reactions following IV bolus
  (25)
• Bleeding is rare in HIT- dont transfuse
  platelets!

Warkentin. Thromb Haemost. 199982439-447
Warkentin. J Crit Illness. 200217(5)172-178.
10
PF-4 ELISAImmunoassay
Anti PF4-Heparin IgG (from patient plasma)
Well containsPF-4 Heparin Complex
Screen phase Patient plasma addedConfirm
phase Patient plasma 100 u/mL UFH
11
Serotonin Release Assay (SRA)A platelet
activation assay
12
HIT is a clinical/pathological diagnosis Labs
studies help corroborate the clinical suspicion
of HIT

• Test Advantages Disadvantages

Immunoassay High sensitivity Low specificity
(false (ELISA) Technically easy positives
common for Rapid turnaround time some
populations) Platelet High specificity Low
sensitivity Aggregation Technique-dependent SRA H
igh sensitivity Technically demanding High
specificity (radioisotopes) (false positives
rare) Not readily available
SRAserotonin-release assay ELISAenzyme-linked
immunosorbent assay. Fabris et al. Arch Pathol
Lab Med. 20001241657-1666 Kelton. Semin
Hematol. 199936(suppl 1)17-21.
13
Iceberg model for HIT
HIT-induced thrombosis 50 of patients with
thrombocytopenia
Activation assay
Isolated HIT
Immuno- assay
Subclinical Antibody
Schematic Iceberg
14
Treatment goals based on pathophysiology and
clinical studies

• Interrupt the immune response
• Discontinue heparin
• Inhibit thrombin actvity
• Treat active thrombosis
• Prevent new thrombosis

15
Direct Thrombin Inhibitors (DTI)
Argatroban
Lepirudin
Bivalirudin
Semisynthetic hirulog
Recombinant hirudin
Synthetic L-arginine derivative
Composition
25-40 min
1.3-3 hrs
40-50 min
Half-life in healthy subjects
Renal
Renal
Hepatic
Elimination
aPTT, ACT
aPTT
aPTT, ACT
Monitoring needed
aPTTactivated partial thromboplastin time
ACTactivated clotting time. Adapted from Chen.
Heart Dis. 20003189-198.
16
Argartroban affects the ProtimeConversion to
oral anticoagulant therapy is an Issue
Initiate warfarin therapy using the expected
daily dose of warfarin while maintaining
Argatroban infusion. A loading dose of
warfarin should not be used.
Measure INR daily
If INR is ?4.0, continue concomitant therapy
If INR is gt4.0, stop Argatroban infusion
Repeat INR 4-6 hours later
If INR is within therapeutic range on warfarin
alone, continue warfarin monotherapy
If INR is below the therapeutic range for
warfarin alone, resume Argatroban therapy
For Argatroban infusion at ?2 mcg/kg/min, the
INR on monotherapy may be estimated from the INR
on cotherapy (see prescribing information). If
the dose of Argatroban gt2 mcg/kg/min, temporarily
reduce to a dose of 2 mcg/kg/min 4-6 hours prior
to measuring the INR.
17
Case 1

• 46 y o female with stage IB Endometrial CA
• TAH BSO, Day 6 DVT/PE ? IV heparin
• Day 9 Leg pain/thrombocytopenia 39,000
• Hematology consult
• Ultrasound ? extension of DVT
• Heparin replaced with Argatroban in fusion
• Labs sent off
• PF4 ELISA positive (OD 1.23, high heparin OD
  0.26)
• PTT was 40 sec, and PT-INR was 1.3 when d/c
  heparin

18
Management of argatroban drip and Conversion to
warfarin Case 1
Orders
Warfarin
Arg-drip
INR
PTT
Platelet
Day
PT/PTT Q 4h
No
2
1.3
40
39K
0
PT/PTT QD
No
3
2.0
50
50K
1
No change
No
3
2.5
55
90K
5
Add warfarin 5 mg Qhs
No
3
2.5
54
150K
6
No change
5 mg
3
4.6
58
200K
10
Stop Arg, check INR in 4 and 8h
5 mg
3
4.7
59
230K
11
D/C Arg
5 mg
No
2.4
32
-
11
19
Summary of HIT

• HIT is a life- and limb-threatening medical
  emergency
• Platelet count monitoring allows early diagnosis
• Laboratory support is crucial
• Specific assay is important to confirm the
  diagnosis
• Monitoring of direct thrombin inhibitors is
  required
• Dose adjusted via PTT or ACT
• Argatroban affects the PT
• makes initiation of warfarin tricky

20
Case 2

• 45 yo female admitted with
• Headache for 1 week, dark urine and lethargy
• PMH Unremarkable, 2 normal pregnancies
• Physical Exam
• Afebrile, BP124/82 mmHg.
• Few petechia on legs, ecchymosis on thigh
• Lab studies
• CBC Hgb 6.8 g/dL, Plt count 15K, 10 Schisto/hpf
• Chemistry LDH 1850 U/L, Creatinine 2.4
• PT-INR 1.2, PTT 31 sec, Fibrinogen 450, DD 189

21
TTP Clinical diagnosis of exclusion

• Classic pentad for recognition
• Laboratory abnormalities
• microangiopathic hemolytic anemia
• thrombocytopenia
• Clinical symptoms (not required for diagnosis)
• renal failure
• mental status changes
• Fever
• Rule out other explainable causes

22
TTP Clinical diagnosis of exclusion

• Microangiopathic Hemolytic Anemia
• Mechanical RBC destruction
• Evidence Schistocytes in peripheral blood

23
TTP Clinical diagnosis of exclusion

• Supporting Laboratory Studies
• RESULT
• CBC with smear schistocytes
• LDH lactate dehydrogenase ???
• Coagulation studies normal
• Haptoglobin ?
• Bilirubin (unconjugated) ?
• Direct Coombs' test negative
• Creatinine, BUN /-?

24
TTP Clinical diagnosis of exclusion

• Diverse Causes of MAHA on blood film
• Thrombotic Microangiopathy (TMA)
• TTP
• Severe hypertension (systolic BP gt 200)
• DIC, sepsis
• HELLP syndrome (preclampsia)
• Metastatic adenocarcinoma
• HIV
• Vasculitis
• Intravascular prosthetic devices
• LV assist device, prothestic valve dysfunction

25
TTP Making the diagnosis in important

• Therapeutic plasma exchange (TPE) is life-saving
  therapy
• Mortality reduced from near 100 to 10
• Most patients will survive acute episode
• but, relapses are common

26
Association of ADAMTS13 with TTP

• 1982 Joel Moake observed presence of
  ultra-large-VWF multimers in circulation of
  patients with relapsing TTP
• He postulated a missing de-polymerase ?
  Persistent ultra-large VWF in plasma ?
  Formation of platelet aggregates ?
  Obstruction of microcirculation ? Organ
  dysfunction ? Clinical presentation TTP

Moake JL J Thromb Haemostas 200421515-1521
27
Milestones Identification of VWF cleaving
protease

• 1996 Tsai and Furlan independently described a
  plasma protease that cleaves normal VWF
• 1997-8 Furlan described 4 patients with
  relapsing TTP who lacked VWF-cleaving protease
• A TTP case with IgG antibody-inhibitor of VWF-CP
  was noted
• 1998 Two large retrospective series evaluated
  VWF-CP levels in patients with TTP and other
  thrombocytopenic syndromes
• Furlan, M. et al. N Engl J Med 19983391578-1584
• Tsai, H.-M. et al. N Engl J Med
  19983391585-1594

28
VWF cleaving protease in TTPFindings of two
retrospective series Furlan et al, Tsai and
Lian, 1998
Tsai, H.-M. et al.N Engl J Med 19983391585-1594

• VWF-CP is generally very low in acquired
  idiopathic TTP
• Inhibitor antibody can be demonstrated in most
  acquired TTP
• VWF-CP levels often come up with TPE-based
  treatment
• VWF-CP is deficiency in congenital TTP
• VWF-CP is normal in HUS and several other
  thrombocytopenic disorders

29
VWF cleaving protease is ADAMTS13 A
Disintegrin-like And Metalloprotease with
ThromboSpondin type 1 motifs, 13

• Cloning showed VWF-CP is an ADAMTS protein

Figure from Lämmle B, et al J Thromb Haemostas
2005 3 1663-1675
30
ADAMTS13 some answers in TTP

• Provides a mechanism of TTP pathogenesis
• Provides a rational for efficacy of TPE
• TPE depletes auto-antibody to ADAMTS13
• Plasma infused during TPE replaces ADAMTS13
• Explains why immune-modulation works
• Does it provide a useful lab test for TTP?

31
ADAMTS13 assay in TTPClinical Utility

• Sensitivity and Specificity is still
  controversial
• No gold standard criteria for a diagnosis of
  TTP
• Distinction of TTP from HUS and other forms of
  TMA is often clinical
• Other practical limitations
• How technically difficult is the assay?
• ADAMTS13 activity assays generally a send-out
  test
• Will assay results available on a clinically
  relevant time frame?
• TPE decision is usually required before the
  results are back
• Test kits using more rapid methods are becoming
  available
• Are there interfering substances that confuse
  result interpretation?

32
Assays of ADAMTS13 activity

• Activity assays
• Based on degradation of VWF (or a VWF fragment)
• Step 1 Proteolysis of substrate by ADAMTS13
• Step 2 Substrate digestion is quantified
• Disappearance of intact VWF by gel-based assay
• Accumulation of a proteolysed degradation product
  (Blot or FRET)
• or
• Decrease of a VWF functional capacity (RCo or CBA
  method)
• Level of detection is 5 of Normal
• Inhibitor assay
• Mix patient plasma with source of ADAMTS13
• Assess residual ADAMTS13 activity
• Report inhibitor units (similar to a coagulation
  factor inhibitor assay)

33
Fluorogenic resonance energy transfer
FRETS-VWF73 Assay
.

• Without ADAMTS13 cleavage, light emitted by Nma
  is absorbed by Dnp-quencher
• Fluorescence is not detected

Kokame, et alBritish Journal of Haematology
2005129 (1), 93-100.
34
Fluorogenic resonance energy transfer
FRETS-VWF73 Assay

• ADAMTS13 cleavage of Tyr-Met bond separates
  Dnp-quencher from Nma emitter
• Fluorescence is detected
• Advantages
• Does not require denaturing VWF
• Assay occurs in real time over 60 min
• Can use kinetic or end-point readings
• Technically adaptable to clinical labs
• Allowing rapid turn-around
• Kits now on market
• Limit of detection 5 of normal

Kokame, et alBritish Journal of Haematology
2005129 (1), 93-100.
35
ADAMTS13 antibody assay

• Qualitative immunoblot-based indicate
• Most inhibitor patients have multiple antibody
  specificities, but antibody to Cysteine-rich/Space
  r domains is very common
• There are some patients with non-inhibitory
  antibody (which may promote ADAMTS13 clearance).
• ELISA assays are emerging for anti-ADAMTS13
• In patients with ADAMTS13 activity lt 10, the
  vast majority had IgG antibody (Reiger M, et al
  Blood 2005)

36
Diagnostic sensitivity at onset of
illnessProportion of patients with severely
deficient ADAMTS-13 activity (Usually defined as
lt5-10)

• Author Design of study Severely
  deficient/total ()
• Vesely et al. 2003 Inception cohort, single
  center 16/48 33
• Peyvandi et al. 2004 Multicenter 48/100 48
• Matsumoto et al. 2004 Multicenter 56/108 52
• Kremer Hovinga. 2004 Multicenter 56/93 60
• Mori et al. 2002 Retrospective? 12/18 66
• Veyradier et al. 2001 Prospective,
  multicenter 47/66 71
• Zheng et al. 2004 Single center,
  prospective 16/20 80
• Furlan et al. 1998 Retrospective,
  multicenter 26/30 86
• Groot et al. 2006 Prospective 24/27 89
• Tsai and Lian 1998 Retrospective 37/37 100

Series collated by Lämmle B, et al J Thromb
Haemostas 200531663-1675
37
Severe ADAMTS13 deficiency is rarein secondary
thrombobotic microangiopathy

• Cancer
• Hematopoietic stem cell transplantation
• Solid organ transplantation
• Preeclampsia
• Systemic infection
• Diarrhea associated HUS

38
Diagnostic specificity in ill patients Other
diseases where low ADAMTS13 activity was observed

• Mild deficiency of ADAMTS13 is common
• Uremia, chronic inflammation, pregnancy,
  post-operatively, liver disease
• Mannucci PM, et al Blood 2001
• Severe deficiency
• Liver disease (Mannucci PM, et al Blood 2001)
• Sepsis-induced DIC (Ono T, et al Blood 2006)
• 17 of 109 patients had ADAMTS13 lt 5 and 51 of
  109 had ADAMTS13lt 20
• Disseminated Malignancy (Oleksowicz et al 1999)
• Mean ADAMTS13 was 4.9 in 10 patients with
  metastatic disease

39
Diagnostic specificity Interfering substances

• Severe hemolysis in sample (Studt JD, et al
  Blood 2005)
• Free hemoglobin inhibits ADAMTS13 activity
• Bilirubin absorbs fluorescence of Nma
• Artifactual reduction of ADAMTS13 activity
  observed by FRET
• Inhibitor in mix assay is bilirubin (not
  autoantibody)

40
Value of ADAMTS13 activity in TTP at
presentationDiagnostic value - 1

• Idiopathic TTP
• Severe deficiency supports a diagnosis of TTP
• Observing an inhibitor probably improves
  specificity
• Not all idiopathic TTP patients present with
  severe deficiency
• Non-low ADAMTS13 level should not ? deny TPE
  therapy!
• Cross-over syndromes
• Systemic Lupus Erythematosis
• Severe deficiency of ADAMTS13 may indicate a role
  for apheresis
• Pregnancy
• ADAMTS13 expected to be adequate in HELLP and
  preeclampsia
• Severe ADAMTS13 deficiency may indicate TTP
  complicating pregnancy

41
Value of ADAMTS13 activity in TTP at
presentationDiagnostic value - 2

• Non-idiopathic TTP
• Vesele S, et al Blood 2003, Raife T, et al
  Transfusion 2004, Zheng XL, et al Blood 2004
• Severe ADAMTS13 deficiency is rare
• Mortality rare is high

42
Prognostic value of ADAMTS13 activity in
idiopathic TTP

• Severe ADAMTS13 deficiency at presentation
• Vesele S, et al Blood 2003, Raife T, et al
  Transfusion 2004, Zheng XL, et al Blood 2004
• High titer inhibitor associated with poorer
  outcome
• A relapsing course was more common
• Unclear if ADAMTS13 level correlates with number
  of TPE required (OK-no, WI-yes)
• In idiopathic TTP, clinical recovery may occur
  despite persistently low ADAMTS13
• Severe ADAMTS13 deficiency after remission
• Nearly all patients with relapsing TTP have
  severe deficiency of ADAMTS13 at the time of
  relapse.
• Should the goal of therapy be suppression of
  autoantibody and restoration of ADAMTS13 level?

43
ADAMTS13 content in TPE replacement fluids

• Essentially equivalent ADAMTS13 content in
• Fresh Frozen Plasma (FFP)
• Cryo-depleted Plasma (CPP)
• Thawed Plasma (Data out to 5 day)

Scott EA, et al Transfusion 200747120-125
44
Case 2

• 45 year old female admitted with
• Headache, thrombocytopenia, renal insufficiency
• Treated with TPE
• Platelets better, retic count came up and
  hemoglobin rose
• Patients mentation became normal
• ADAMTS13 was lt 5, inhibitor 3
• This patient had TTP

45
Summary ADAMTS13 assay

• Is lab assay of ADAMTS13 clinically useful? YES
• A very low level (with inhibitor or Ab) supports
  a dx of TTP
• A low level may argue for a trial of TPE in
  cross-over syndromes
• A level gt 10 does not exclude TTP
• ADAMTS13 level should not drive TPE schedule at
  current time
• Is ADAMTS13 prognostic of disease course in TTP?
  YES
• Low activity at presentation or in remission is a
  risk factor for relapse
• Does ADAMTS13 explain it all? No
• There is much is yet to be learned.

46
Case 3 An 2 year old boy with nose bleeds and
a long bleeding time

• Nose bleeds occur during day or night.
• Bleeding occurred after circumcision.
• Bruising began once he became mobile.
• Mother had menorrhagia with anemia
• PTT, PT, and platelets all normal
• But PFA-100 times were gt 250 sec

47
Functions of VWF

• Supports platelet adhesion and activation at
  sites of vascular injury
• VWF binds extravascular collagen
• Platelets adhere to bound VWF
• Adherent platelets become activated
• Supports coagulation mechanism
• VWF protects Factor VIII in circulation
• VWF co-localizes F-VIII at sites of vascular
  injury

48
Utility of PFA-100 in VWD

• Evaluation of patients with diagnosed VWD1
• Sensitivity of PFA 95, Sensitivity of BT 65
• Screening of Patients with Bleeding Symptoms2-4
• Sensitivity 62-80
• Specificity 84-89

1. Fressinaud E, et. al Blood 1998911325-13312
. Posan E, et al Thromb Haemostas
200390483-903. Quiroga T, et al J Thromb
Haemostas 20042892-8984. Phillips C et al
Obstet Gynecol 200510561-66
49
To diagnose VWD

• You need to specifically test VWF

50
Pathophysiologic Classification of von
Willebrand Disease

• Inherited Quantitative Defects
• Type 1 Low VWF protein level
• Type 3 Absence of VWF
• Inherited Qualitative Defects
• Type 2
• Poor platelet-binding function
• Due to small multimers Type 2A
• Normal size multimers (abn A1 loop) Type 2M
• Accentuated platelet-binding function
• Abnormal A1 loop of VWF Type 2B
• Abnormal platelet GP Ib Platelet type
• Decreased Factor VIII Binding Type 2N
• Acquired VWD

51
Lab analysis for Von Willebrand Disease

• Quantitative assays
• VWF activity Functional ability of VWF to bind
  and agglutinate platelets
• Ristocetin cofactor activity
• VWF antigen Amount of VWF protein present
• F-VIII by coagulation assay
• Discriminating studies for VWD subtype
• Multimer analysis (size of VWF molecules)
• Ristocetin-induced platelet aggregation
• Special binding assays
• Platelet, factor VIII and collagen binding assays
• Genetic analysis

52
Inherited Quantitative Defects

• Type 3 VWD
• Essentially no detectable VWF
• VWF antigen and VWF activity are undetectable
• ? mucosal bleeds
• F-VIIIC is low
• ? deep tissue bleeds
• Rare autosomal recessive inheritance
• Type 1 VWD
• Bleeding is attributable to partial VWF
  deficiency
• VWFAg and VWFRCo are similarly low
• Factor VIII level is mildly depressed or normal
• Common 80 of cases of VWD
• Autosomal dominant with variable penetrance
• Note
• Inflammation, stress, hormone, etc. affect VWF
  levels

53
Pathophysiologic Classificationof von Willebrand
Disease

• Inherited Quantitative Defects
• Type 1 Low VWF protein level
• Type 3 Absence of VWF
• Inherited Qualitative Defects
• Type 2
• Poor platelet-binding function
• Due to small multimers Type 2A
• Normal size multimers (abn A1 loop) Type 2M
• Accentuated platelet-binding function
• Abnormal A1 loop of VWF Type 2B
• Abnormal platelet GP Ib Platelet type
• Decreased Factor VIII Binding Type 2N
• Acquired VWD

54
Functional RatioVWFRCo / VWFAg
Cut-off (ratio 0.5)
Adcock DM, et al Semin Thromb Hemost
200632472-479
55
VWF Multimer Analysissize
NP
VSD
VSD
TTP
type 1
type 3
type 2A
type 2B
56
Type 2A VWDPoor platelet-binding function Small
Size

• Quantitative VWF assays are usually low
• VWFAg gt VWFRCo
• Only small multimers present
• Absence of large VWF multimers
• Mechanisms
• Group 1 abnormal intracellular
  assembly/transport
• Group 2 accelerated proteolysis
  (tyr1605-met1606)
• Autosomal dominant Point mutations in VWF gene

57
VWF MultimersType 2ALoss of highest multimers
spectrum
NP
NP
Type 2A-1
Type 2A-1
Type 2A-2
Type 2A
58
Type 2B VWD Increased platelet-binding function
Clear VWF/platelet

• Slightly low VWFAg, lower VWFRistocetin
  Cofactor
• Loss of highest molecular weight multimers
• Platelet count is low
• Qualitative variant with increased affinity for
  GP Ib
• Gain in function
• Enhanced clearance of VWF and platelets from
  circulation
• Platelet aggregation with low dose ristocetin
• Autosomal dominant pattern of inheritance
• Mutations in A1 domain that binds platelet GP Ib

59
Spectrum of VWF Multimers Type 2B versus
Type 2A
NP
NP
NP
NP
Type 2B
Type 2B
Type 2A-1
Type 2A-1
Type 2A-2
Type 2A
60
Ristocetin-induced Platelet Aggregation
Ristocetin 0.5 mg/ml
Ristocetin 1.5 mg/ml
Risto 0.5
Risto 0.5
Risto 1.5
Risto 1.5
Control
Type 2B Von Willebrand disease(Platelet-type VWD
gives similar pattern)
61
Platelet Type VWD Increased platelet-binding
function Clear VWF/platelet

• Same clinical characteristics as type 2B
• Gain in function due to defective platelet
  GP-Ib receptor
• Autosomal dominant pattern of inheritance
• Genetic defect in platelet GP Iba
• Treatment of bleeding Platelet transfusion

62
VWF Binding to Target Platelets at Ristocetin
0.3 mg/ml
I125

Type 1
Normal
Platelet Type
Type 2A
Type 2B
63
VWF 178 kb gene 52 exons
protein
propeptide (VW AgII)
mature von Willebrand factor (VWF)
aa.
842
1
741
2050
1
843
1000
500
1500
500
A1
A3
S
S
D1 D2
D3 A1 A2
A3 D4 B1B2 B3
C1 C2
N
C
RGDS
FVIII
GPIb
collagen
heparin
GPIIb/IIIa
heparin
collagen
furin cleavage site
cDNA
0
1
2
3
4
5
6
7
8
kB
type 2A-1 VWD
type 2A-2 VWD
type 2A-1 VWD
type 2B VWD
type 2N VWD
?RRM
type 2M VWD
64
Case 3. An 2 year old boy with nose bleeds and
a long bleeding time

• Nose bleeds occur during day or night.
• Bleeding occurred after circumcision.
• Bruising began once he became mobile.
• Mother had menorrhagia with anemia
• PTT, PT, and platelets all normal
• PFA-100 closure times were gt 250 sec

65
3. vWF Results

• vWF activity 16
• tests vWFplatelet interaction
• vWF antigen 50
• quantitates vWF protein
• vWF multimers Only smallest bands
• measures the size of the vWF
• Factor VIII 45
• measures vWFFVIII interaction

Dx Type 2A vWD
66
Summary Special assays for VWD

• Screening tests dont necessarily exclude VWD
• If bleeding is out of proportion to the assay
  results, or if the VWF ristocetin cofactor level
  does not line up with the VWF antigen, more
  testing may be indicated.
• There are many special VWF assays
• Multimer assay is a good place to start.
• Occasionally genetic sequence testing is required
  to make a diagnosis.