The Clinical Use and Immunologic Impact of Thrombin in …library.corporate-ir.net/library/13/130/130168/items/176476... · The Clinical Use and Immunologic Impact of Thrombin in

The Clinical Use and Immunologic The Clinical Use and Immunologic Impact of Thrombin in SurgeryImpact of Thrombin in Surgery

Jeffrey H. Lawson, M.D., Ph.D.Departments of Surgery and Pathology

Duke University Medical CenterDurham, North Carolina

Bovine ThrombinHuman Thrombin

and Who Cares

116.3Protein bands above the molecular weight of α-Thrombin

{

γ-Thrombin

(presumed)

α-Thrombin

β-ThrombinThrombin B-chain

200

66.3

55.2

36.5

14

6

BT1 BT2 rhTKDa

BT1: Thrombin-JMI® 20 U, Lot 1

BT2: Thrombin-JMI® 20 U, Lot 2

rhT: rhThrombin 20 U

Thrombin - B4 chain

α-Thrombin

α-Thrombin with truncated B-chain

Thrombin B-chain

β-Thrombin

The Many Faces of Thrombin

What is Topical Bovine Thrombin?

Bovine Thrombin Preparations• Are used in a variety of surgical procedures

including, but not limited to:– Cardiovascular Surgery– Vascular Surgery– Neurologic Surgery– Orthopedic Surgery– General Surgery– Gynecological Surgery

• Estimated that more than 500,000 Americans are exposed each year

• More than 100 reports in the worlds literature of adverse events related to bovine thrombin exposure in humans

Clinical Reports of Patients with Clinical Reports of Patients with Major Complications from AntibodiesMajor Complications from Antibodies

FDA FDA ““GrandfathersGrandfathers”” Bovine Bovine Thrombin for Commercial Thrombin for Commercial ProductionProduction

First Clinical Use of Thrombin in HumansFirst Clinical Use of Thrombin in Humans

19401940 19501950 19601960 19701970 19801980 2000200019901990 20102010

YEARYEAR

-200

-300

-100

Patient Profile

• 68 year old WM• Myocardial Infarction - 1970• MVR and CABG x 1 - 1986• Repeat MVR - 1987

– 10,000 units of Bovine Topical Thrombin• Repeat MVR - 1988

– 10,000 units of Bovine Topical Thrombin

Lawson et al., Blood, 1990

Patient LabsTest Name Patient

Value Normal Range

Prothrombin time 17s 11-13s APTT 34s 22-33s Thrombin time (Bovine)

>150s 16-20s*

Thrombin time (Human)

35s 16-20s*

Platelet count 363,000/µL 130-430,000/µL Fibrinogen 465mg/dL 170-330mg/dL D dimer 0.2µg/mL <0.2µg/mL FDP 10µg/mL <10µg/mL

*Thrombin concentration is adjusted to give a clotting time of 16 to 20 seconds in control plasma, normal is considered ± 3 seconds of control plasma.


Isolation of Anti-thrombin Antibody from Patient Plasma





“Important Drug Warning”

“The use of topical bovine thrombin preparations has occasionally been associated with abnormalities in hemostasis…”

“Patients with autoantibodies to bovine thrombin

preparations should not be re-exposed to these

products.”

Immunologic Impact and Clinical Outcomes Following Surgical Exposure

to Bovine ThrombinThomas Ortel, Meg Mercer, Elizabeth Thames, Karen

Moore, and Jeffrey H. Lawson

Duke University Medical CenterDurham, NC

Ortel et al. Annals of Surgery, 233(1), 88-96, 2001

Study DesignPatient Population Number of Individuals

Normal Individuals (CONTROL) 50CABG (Primary) 100 Valve Surgery Patients (Primary) 50Valve Surgery Patients (Redo) 25

Total 225


Blood Sample Collection• Preoperative• 24 - 48 hours Postoperative• 4-7 days Postoperative• 4-8 weeks Postoperative

Patient Outcomes• Hemorrhage• Thrombosis• Infection• Wound Healing• Transfusion• Death


Screening Laboratory Assays• PT, aPTT, and TCTImmunoassays:• Anti- Bovine Thrombin

Preparation IgG• Anti- Bovine Thrombin IgG• Anti- Bovine Prothrombin IgG• Anti- Bovine factor V IgG• Anti- Bovine factor Va IgG• Anti- Human Thrombin IgG• Anti- Human Prothrombin IgG• Anti- Human factor V IgG

ResultsPatient Population Number of IndividualsTotal Enrolled 226

Incomplete Follow-up 16Deaths 9

Total Completed 201Normal Individuals 50CABG (Primary) 90Valve Surgery (Primary) 34Redo Sternotomy 27



Pre and Post Operative Response to Bovine Antigens Following Bovine Thrombin Exposure

Response to Bovine Antigens

TBT bFV bFVa bFII bFIIa0

10

20

30

40

50

60

70

80

90

100Primary Exposure Prior Exposure All Exposed

Bovine Coagulation Factor

Perc

ent D

evel

opin

g Ig

G A

ntib

odie

s

Anti-Bovine

?


The Phylogeny of αGal

Mammals

CowsMice

PigsNew World Monkeys

αGal

Old World MonkeysApes

HumansαGal

αGal not expressedαGal expressed

Natural Anti-αGalAntibodies

Contamination - Multiple Proteins and αGal

Kd

218-

125-

78-

44-

32-

7.7-

Thrombin TBT

αGal

Schoenecker et al. J. Clin. Immun. 20(6):434-44,2000.

Increase in Elicited IgG

Unexposed Exposed

-10

-1

1

10

100

1000

Exposure to Bovine Thrombin

Cha

nge

In T

iter

?Schoenecker et al. J. Clin. Immun. 20(6):434-44,2000.

Conclusions - αGal• Topical Bovine thrombin contains αGal.

• Patients have xenoreactive natural antibodies which are specific for the bovine thrombin preparation proteins.

• Levels of elicited IgG anti- αGal are significantly increased 6 weeks following exposure to bovine thrombin preparations.

αGal

Interesting Xeno-Immunology

But what does that have to do with surgery and my patients?

The BlueBlue--Devil is in the Details

Response to Human Antigens

hFV hFII hFIIa0

10

20

30

40

50

60

70

80

90

100Primary Exposure Prior Exposure All Exposed

Human Coagulation Factor

Perc

ent D

evel

opin

g Ig

G A

ntib

odie

s

Anti-Human?

Abnormal Coagulation Studies

PT aPTT TCT Total0

10

20

30

40

50Primary ExposurePrior Exposure

Coagulation Test

Perc

ent A

bnor

mal

Antibodies ABNORMAL

CLOTTING

TESTS?


Pre and Post Operative Response to Bovine Antigens Following Bovine Thrombin Exposure

Preoperative Antibody Response to Bovine Antigens

With (+) and Without (-) Documented Prior Exposure

With (+) Without (-)

10%

Adverse Events Related to Pre-operative Antibody Levels

Multivariant Analysis of Adverse Events

0

25

50

75

100

Patient Group

Perc

ent P

oor

Out

com

es A

fter

Surg

ery Antibody Negative

Antibody Positive

Outcomes

46/136

(34%)

11/15

(73%)

Antibodies

P=0.0042

OUTCOME

“Re-exposure”

? ?

Bovine Thrombin “Black Box” Warning

What is the Cost of Exposure to Antibody Positive Patients?

PEPCI

Conclusions1. Bovine topical thrombin is highly immunogenic

when used in the setting of cardiovascular surgery.

2. Thirty percent of patients exposed to bovine thrombin develop cross-reactive antibodies with human blood coagulation factors.

3. Thirty percent of patients with anti-clotting factor antibodies develop abnormal blood coagulation tests.

PEPCI

Conclusions4. Anticoagulation monitoring in patients with anti-

clotting factor antibodies is problematic.

5. Patients with multiple elevated antibodies prior to surgery are more likely to sustain adverse clinical outcomes.

Adjusted Odds Ratio: 5.4095% Confidence Interval: 1.54, 18.8

6. Patients with multiple elevated antibodies prior to surgery appear to have increased cost associated with the procedure which is independent of preoperative comorbid conditions.

Vascular Access Patients with circulating antibodies to Bovine Thrombin Preparations

50% of patients with PTFE grafts have these antibodies

Greater than 80% of patients with a history of graft thrombosis have elevated antibody levels to bovine thrombin reagents

Anticardiolipin Antibody Formation 6 Weeks after CABG

Pre-op Post-op0

20

40

60

80

100A.

Ant

icar

diol

ipin

ant

ibod

y Ig

G ti

ter (

GP

L)

Pre-op Post-op0

20

40

60

80

100B.

Ant

icar

diol

ipin

ant

ibod

y Ig

M ti

ter (

MP

L)

Pre-Op Post-Op0

20

40

60

80

100C.

Ant

icar

diol

ipin

ant

ibod

y Ig

G ti

ter (

GP

L)

Bovine Thrombin Exposure CABG CABG no Thrombin

Su et al. Journal of Clinical and Laboratory Medicine, 139(6):349-56, 2002.

Exposure of Mice to Bovine Thrombin

Antibodies against αGal and Bovine Thrombin (TGEN)

Schoenecker et al. American Journal of Pathology, 159(5),1957-69, 2001

Development of Anti-Clotting Factor Autoantibodies and Abnormal Coagulation Tests

SALINE TGEN

Cha

nge

in A

bsor

banc

e


0

1

2

3

4

5

aPTT

/PT

Rat

io

P=0.0159

Control Saline TGENUnexposed Exposed

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Cha

nge

in A

bsor

btio

n

Unexposed Exposed

Antibodies against double stranded DNA and Cardiolipin


Thrombin Control


So What about Floseal??

Lawson et al. Ann Thorac Surg, 79:1038-40, 2005

Lawson et al. Ann Thorac Surg, 79:1038-40, 2005

The Immunology of Human Fibrin Sealant and Human

Thrombin

Immunogenicity of rhThrombin

1/37

2.7 (0.1–14)

0/21

0 (0–16)% (95% CI)

Any rhThrombin

N=38Placebo Only

N=22

Anti-rhThrombin Antibody Incidence

Mouse Exposure to Thrombins from Various Species

Case Study (II)• 76 year old male admitted for Abdominal Aortic

Aneurysm Repair

Past Surgical History: 1971 Laryngectomy1990 Coronary Artery Bypass Grafting x 4 Vessels1996 Rotator Cuff Repair1998 Left Radical Nephrectomy

-Thrombin spray documented in operative report1999 Right Radical Nephrectomy

-Patient readmitted for right flank hematomaHistory of transient ischemic cerebrovascular attacks-taking

Coumadin

Case Study (II)Hospital Course:• 12/20/2000 Abdominal Aortic Aneurysm and Bilateral

Iliac Artery Aneurysm Repair• Intraoperatively received 20,000u Thrombin Spray and

Flo-Seal (10,000u Thrombin) as documented in the perioperative nursing record

• Returned to the ICU a few days postoperatively for increased SOB, episodes of epistaxis, and difficulty breathing requiring re-intubation and mechanical ventilation.

• Patient developed profound coagulapathy and noted as having a factor V inhibitor

Antibodies ResponseBovine Antigens - Post-OP Day 5

Antibodies ResponseHuman Antigens - Post OP Day 5

Recent Case Study Con’t)Treatment:• Received copious amounts of blood and factor

replacement. • Continued to have increasing abdominal distension and

hemodynamically unstable.• CT scan suggested of intraperitoneal bleed • 1/7/2001 Received Immune Globulin 10% (Gamimune-N)

-IVIG• 1/8/2001 Take back to the OR for exploratory laparotomy.

Removed 3 liters of mostly clotted blood.• Prolonged ICU course with Ventilator Dependence• DC’d (2/12/2001) to a nursing facility that takes care of

ventilator dependent patients

Recent Case Study (Con’t)

Discharge Diagnosis

“Acquired coagulopathy secondary to factor V inhibitor, presumed secondary topical bovine thrombin.”

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The Clinical Use and Immunologic Impact of Thrombin in …library.corporate-ir.net/library/13/130/130168/items/176476... · The Clinical Use and Immunologic Impact of Thrombin in