Inhibitor of the Thrombin Time in Systemic Amyloidosis

Inhibitor of the Thrombin Time in Systemic Amyloidosis

From www.bloodjournal.org by guest on October 12, 2016. For personal use only.
Inhibitor of the Thrombin Time in Systemic Amyloidosis:
A Common Coagulation Abnormality
By Dennis A. Gastineau, Morie A. Gertz, Todd M. Daniels, Robert A. Kyle, and E.J. Walter Bowie
Patients with primary systemic amyloidosis (AL) often experience bleeding, and we report a newly recognized coagulation abnormality in AL. Of 103 patients with primary systemic
AL studied over 2 years, 41 had prolongationof the thrombin
time (range, 25 to 46 seconds; normal, <22 seconds) and
reptilase time (range, 17 to 39 seconds; normal, 14 to 16
seconds). The fibrinogen from the plasma of 36 patients was
precipitated by p-alanine and diluted to a concentration of
approximately200 mg/dL. The thrombin times of the precipitated fibrinogens were normal in 34 patients, implying that
an inhibitor was responsible for the abnormal tests. The
addition of patient fibrinogen-free plasma to normal plasma
prolonged the thrombin times, and this result confirmed the
presence of an inhibitor. The inhibitor is more likely to be
present in patients with nephrotic syndrome (20 of our
patients) and congestive heart failure (six). A circulating
monoclonal protein (24 patients), the presence of amyloid
liver involvement (eight), and the presence of amyloid neuropathy (nine) were not predisposing factors. Only one patient had deficiency of factor x. We conclude that inhibition
of fibrinogen conversion to a fibrin clot rather than dysfibrinogenemia is the cause of the prolonged thrombin time in
primary systemic AL.
o 1991 by The American Society of Hematology.
P
gauze. The sample was rejected if visible clots were present.
Routine coagulation assays, including thrombin time, prothrombin
time (Thromboplastin C; Dade, Miami, FL), activated partial
thromboplastin time (Organon Technica, Durham, NC), reptilase
time, protamine gel, and fibrinogen determination, were performed as previously described.”
Precipitation offibrinogen. One milliliter of 6.0 mol/L p-alanine
was added to 2.0 mL plasma in plastic test tubes. The sample was
gently vortexed and placed on melting ice for 30 minutes. It then
was centrifuged for 20 minutes at 4°C at 8,700g. The supernatant
was discarded. The precipitate was rinsed once with 0.017 mol/L
imidazole, 0.10 N NaC1, 0.05 mollL aminocaproic acid, pH 7.4:
This buffered saline, 1.1 mL, was added to the precipitate, which
was then warmed to 35°C for 5 minutes and dissolved. Fibrinogen
composed approximately 50% of the dissolved protein, based on
residual absorbance after clotting 1 mL protein solution with 10
units bovine thrombin (n = 5). Using an extinction coefficient of
1.64” for 0.1% (100 mg/dL) solution of fibrinogen at 280 nm, and
because 50% of total absorbance was previously shown to be
fibrinogen, the protein solution was diluted with buffered saline to
6.6 absorbance units. This dilution yields approximately 200 mg/dL
fibrinogen. Bovine thrombin (Parke-Davis, division of WarnerLambert, Morris Plains, NJ) was diluted in buffered saline to give a
control PPP thrombin time of 20 2 0.5 seconds ( 2 2 SD) (approximately 15 IJ.Lof thrombin in 10 mLof buffered saline).”,”Duplicate
thrombin times were determined on each PPP and diluted fibrinogen isolate. The precision of duplicate thrombin times performed
in the assays was t0.32 second (n = 25).
Depletion offibrinogen. Normal plasma and amyloid PPP were
depleted of fibrinogen with 0.5% bentonite.” Fifty microliters of a
100 mg/mL suspension of bentonite (Sigma, St Louis, MO) in
buffered saline was added to 1 mL of plasma and placed on a
rocking platform for 30 minutes at room temperature. Samples
were centrifuged at 1,OOOg for 15 minutes and the supernatants
were tested for the presence of inhibitor. Equal volumes of normal
supernatant or amyloid supernatant were mixed with normal
ATIENTS WITH PRIMARY systemic amyloidosis
(AL) may experience bleeding. Much attention has
been focused on the presence of abnormal prothrombin
times and associated factor X deficiencies,’ but we have
recognized a far more common coagulation abnormality in
amyloidosis- a prolonged thrombin time. Prolonged thrombin times associated with acquired factor X deficiency have
been described in only a few
Prolonged thrombin times and normal reptilase times
have been reported in patients with another related dysproteinemia (multiple myeloma) and a heparan sulfate proteoglycan anticoagulant has been isolated and characterized‘
Abnormal and inhibited thrombin times associated with
abnormal reptilase times often are interpreted as representing dysfibrinogenemia, as frequently seen in patients with
severe liver di~ease.~.’
We examined the coagulation studies of 41 patients with
AL and investigated the nature of the prolongation of the
thrombin time by partially purifying the fibrinogen of 36 of
these patients.
SELECTION OF PATIENTS
Patients seen in the Dysproteinemia Clinic at the Mayo
Clinic are routinely screened for coagulation abnormalities,
most often for factor X deficiency. Over 2 years, 103
patients with primary systemic AL were studied in the
coagulation laboratory. All of these patients had thrombin
times determined. Forty-one patients with a prolonged
thrombin time ( > 5 seconds in excess of control) and the
diagnosis of AL who were seen between April 8,1986, and
March 28, 1988, were studied. A sufficient sample was
available in 36 patients, and the fibrinogen was isolated
from the plasma. The fibrinogen of 25 controls also was
isolated.
MATERIALS AND METHODS
Collection and handling of specimen. Venous blood was collected with a plastic syringe and dispensed into a plastic vial
containing trisodium citrate and aprotinin (final concentration
0.38% and 260 KIU/mL, respectively) (aprotinin from Trasylol,
Mobay Chemical Corporation, New York, NY).Blood was centrifuged at 4°C at 1,700g for 10 minutes. Platelet-poor plasma (PPP)
was removed and placed at 4°C if tested the same day or frozen at
-20°C for later testing. The cellular fraction was strained through
Blood, Vol77, No 12 (June 15), 1991: pp 2637-2640
From the Division of Hematology and Internal Medicine, Mayo
Clinic and Mayo Foundation, Rochester, MN.
Submitted February 26,1990; accepted February 11, 1991.
Address reprint requests to Dennis A . Gastineau, MD, Mayo Clinic,
200 First St SU: Rochester, MN 55905.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Socieb of Hematology.
0006-4971I91j77I2-0001$3.O0jO
2637
From www.bloodjournal.org by guest on October 12, 2016. For personal use only.
GASTlNEAU ET AL
2638
plasma and allowed to incubate at room temperature for 30
minutes. and thrombin times were determined.
Plasma was depleted of fibrinogen by heating in a 56°C waterbath for 4.5 minutes. Samples were centrifuged and tested for the
presence of inhibitor as described above.
The mean thrombin time of the normal PPP was
fnrep”ion.
20.9 seconds (2 SD range, 17.7 to 24.1 seconds; 1 SD.1.6 seconds;
n = 35). The mean thrombin time of the normal isolated fibrinogen
was 17.4 seconds (2 SD range, 14.9 to 19.9 seconds; 1 SD. 1.25
seconds; n = 35).
A normal result is a purified fibrinogen thrombin time less than
or equal to the normal PPP thrombin time (generally 18 to 21
seconds). A normal isolated fibrinogen thrombin time and prolonged PPP thrombin time are consistent with the presence of an
inhibitor. The prolongation of both clotting times is consistent with
a dysfibrinogcnemia.
RESULTS
The fibrinogcn from the plasma of 36 patients with
abnormal thrombin timcs was prccipitatcd by p-alaninc and
diluted to a conccntration of approximately200 mg/dL. Thc
thrombin times of the prccipitatcd fibrinogcns wcrc normal
in 34 of thc 36 patients. implying thc presence of an
inhibitor.
Thrcc patientswith known dysfibrinogcnemia (one plasma
samplc was kindly provided by Dr Douglas Triplctt, Muncic, IN) wcrc studicd. The isolated fibrinogcns of thcsc
patients produccd prolongcd thrombin times (Fig 1). All
wcrc substantially morc prolongcd than the only two
amyloid-associatcd fibrinogcns that did not correct to the
normal range.
Normal and amyloid PPPs were depleted of fibrinogen
using 0.5% bcntonitc or heating to 56°C for 4.5 minutcs.
Aftcr ccntrifugation, the supcrnatants wcrc mixcd with
normal PPP and thrombin timcs wcrc dctcrmincd. Supematant from thc thrcc amyloid plasmas prolongcd the thrombin timc (Tablc 1).
The thrombin timc waq the only coagulation test result
that was consistcntly abnormal: prolongcd in 36 of thc 41
paticnts studicd (Tablc 2). Clottablc fibrinogen lcvcls
(biuret method) generally were clcvatcd (range, 210 to
1,190 mg/dL; mcdian, 636 mg/dL) in paticnts whosc thrombin timc was prolongcd. No paticnt had hypofibrinogcn-
.-$ 3 0
L
C
z 2 0
P
”
N m l
Dysfibrinwen
Amyloid
Fig 1. Thrombin tinm of PPP 10)and hoisted IIMnogen ( 0 )in
nomuh (n = 25). patients with dyrfibrinogenmla (n = 3). and patients with AI. (n = 36).
T a b 1. MiringStudha to D.monrtnt.inhibitor of Thrombin Tim
Thrombin
Time
IPPP, wc)
Diegnoria
Normal
Thrombin
Time
Bentonile.
(isolated
Suparmtent +
fibrinogen. see) Normal PPP
Heal-Wstedt
Supemecsnt +
lnonnnl.
20.9 2 3.2)
(normal,
17.4 2 1.251
(1:l)
Norms1PF’P
(1:l)
(wc)
IS=)
18
15
22
26
64
50
26
58
16
18
17
21
41
32
25
32
29
37
30
34
Nonamyloid
(DC)
Amyloid 1
Amyloid 2
Amyloid 3
Abbreviation: DIC, disseminated intravascularcoagulation.
*Plasma was depleted of fibrinogen using 0.5% bentonite and then
mixed and centrifuged. Equal volumes of supernatant and normal PPP
were incubated 30 minutes at room temperature before determining
thrombin time.
tPlasma depleted of fibrinogen by heating in 56T water bath for 4.5
minutes.
cmia. Consistcntly ncgativc protamine gel tests indicatcd
the absence of active disseminated intravascular coagulation. The rcptilasc timc was consistcntly clcvatcd (rangc, 17
to 39 seconds; median, 24 scconds; control, 15 seconds) in
direct proportion to thc prolongation of thc thrombin timc,
suggesting that a heparin-likc inhibitor was not present.
The clinical abnormalitics arc listcd in Tablc 3. Thc most
common findings wcrc abnormal cchocardiograms and
nephrotic syndromc. Although thcsc wcrc thc most common, no corrclation with thc dcgrcc of prolongation of thc
thrombin timc was found.
A circulating monoclonal protein was seen in 24 of the
patients. The results of immunoelectrophoresis arc shown
in Tabk 4. The scrum monoclonal protcin was gencrally
small in amount and cxcccdcd 1 g/dL in only ninc of the
paticnts and cxcccdcd 2 g/dL in only one. Ninc paticnts had
a scrum monoclonal pmtcin and did not havc associated
congestive hcart failurc or ncphrotic syndromc. Thc thrombin timc in thcsc ninc paticnts rangcd from 19.7 to 41.8
scconds (median, 22.3 scconds). In thc fivc patients without
a monoclonal scrum protcin and without hcart failure or
nephrotic syndromc. thc thrombin timc rangcd from 20 to
26.3 scconds (mcdian, 23.4 scconds). Only onc paticnt had
a prolongcd prothrombin timc (22.2 seconds), and this
paticnt had a factor X lcvcl of 6%. Thc factor X lcvcls wcrc
not dcprcssed bclow 40% in any of thc othcr patients.
Figurc 1 shows the results of thc thrombin timc bcforc
and aftcr thc fibrinogcn was purified. Of thc 36 paticnts
whosc thrombin timc was prolongcd 2 scconds grcatcr than
control, thc thrombin timc normalized in 34. Thc thrombin
timc was shortcncd a mcdian of 8 scconds in thc 36 whosc
thrombin timc was abnormal. In a control group of normals,
purification of the fibrinogcn rcsultcd in a median shortcning of the thrombin timc by 4 seconds.
Ncithcr incrcasing thc protcin canccntration with bovinc
scrum albumin nor adding fibrin split products (Dadc)
rcsultcd in prolongation of the thrombin timc (data not
shown).
From www.bloodjournal.org by guest on October 12, 2016. For personal use only.
2639
THROMBIN TIME INHIBITOR IN AMYLOIDOSIS
Table 2. LaboratoryTest Results in 41 Patients With AL
Patient Values
Laboratory
Characteristic
Median
Normal
Value
No. Abnormal
Results
Tested
19.7-42
17-39
27.3
24.3
20.9 k 3.2
14-16
10.9-22.2
6-184
0.2-3.2
11.7
85
0.8
10.3-12.3
70-140
Absent
36
38
9
1
24
41
38
34
41
41
Range
Thrombin time (sec)
Reptilase time (sec)
Prothrombin time (sec)
Factor X (%)
Serum M protein (g/dL)
DISCUSSION
The thrombin time can be prolonged for several reasons,
including (1) the presence of heparin, ( 2 ) hypofibrinogenemia, (3) dysfibrinogenemia, (4) circulating anticoagulants,
and (5) the presence of fibrin or fibrinogen split products.
Differentiation of most of these causes is not difficult
through the quantitative determination of the fibrinogen
value and of the reptilase time to exclude heparin-type
anticoagulants and through assays for fibrin degradation
products and fibrin monomers.
However, generally dysfibrinogenemia is diagnosed
through exclusion of other causes because no simple in vitro
test exists to confirm the presence of a structurally abnormal fibrinogen. The cause of prolonged thrombin times in
AL has been previously attributed to the presence of an
abnormal fibrinogen. These studies report prolongation of
prothrombin, activated partial thromboplastin, or thrombin
times, but no mixing studies or purification of fibrinogen
was reported.13,'4
Bleeding in amyloidosisis multifactorial. Vascular infiltration of amyloid fibrils disrupts vascular integrity and has
been associated with recurrent gastrointestinal hemorrhage.15 Coagulation factor deficiencies have been well
established, and the apparent ability of amyloid fibrils to
Only one of our 41
bind factor X has been
patients had a prothrombin time greater than 2 seconds
beyond control, and that patient had a factor X level of 6%;
thus, factor X deficiency is rarely associated with an
abnormal thrombin time. Severe liver involvement by amyloid causes deficiencies of many hepatically synthesized
clotting factors and can imitate disseminated intravascular
coagulation and fibrinolysis through failure of hepatic
clearance of fibrin split products. Prolongation of the
thrombin time in severe nonamyloid liver disease is due to
abnormal fibrinogen sialic acid content resulting in abnormal fibrin monomer polymerization. Increased sialyltrans-
No.
ferase activity produces a fibrinogen similar to fetal fibrinogen.6s7Adding excess calcium characteristically shortens the
thrombin time.
The presence of a serum monoclonal protein (usually
associated with multiple myeloma) has been associated
with heparin or heparin-like circulating anticoagulants?
The prolongation of the thrombin time can be partially
reversed with protamine. The thrombin time may also be
shortened by the effects of chemotherapy, as we recently
observed in a patient with a heparan-like inhibitor. These
patients may experience severe bleeding, especially from
the gastrointestinal tract."
Our data suggest that the monoclonal protein in our
patients did not cause the abnormal thrombin time because
the thrombin times were not different between those
amyloid patients with and those without a serum monoclonal protein. The thrombin time abnormality did not have
any effect on the likelihood of clinical bleeding or thrombosis.
Hypofibrinogenemia may result from defective production of fibrinogen, as may be seen in severe liver disease?"
Total fibrinogen concentrations were preserved in our
patients despite significant hepatomegaly in six. In fact, all
patients tested had fibrinogen values greater than 200
mg/dL.
Circulating anticoagulants of the lupus-type were not
specifically sought, and although some patients with monoclonal proteins have a lupus-type activity, these patients are
rare, comprising only 10 of 219 such patients in our
laboratory. None of these patients had amyloidosis.21
Butler and Baldwin' described a patient with factor X
deficiency who also had a prolonged thrombin time, and in
a review of the literature they found that eight of 14
patients had either a thrombin time greater than 1.5 times
control or a prolonged thrombin time that was not corrected in vitro when mixed with normal plasma. They
postulated, as have others: that inhibitors may be present,
Table 3. Clinical Data From 41 PatientsWith Amyloidosis
~
Clinical Characteristic
No. of
Patients
Female
Male
Congestive heart failure
Abnormal echocardiogram
Amyloid neuropathy
Hepatomegaly ( > 2 cm below costal margin)
Nephrotic syndrome ( > 3 g proteid24 h)
Renal insufficiency (creatinine, > 1.5 mg/dL)
15
26
6
19
9
8
20
8
Age range was 42 to 84 years (median, 52 years).
Table 4. Ig Subclasses in 24 Patients With a Serum
Monoclonal Protein
Subclass
No. of Patients
IgG A
GK
Ah
Dh
Free K only
Free A only
9
4
3
1
3
4
7:17
K:
A
From www.bloodjournal.org by guest on October 12, 2016. For personal use only.
GASTINEAU ET AL
2640
but they did not demonstrate direct evidence of inhibition
or the presence of normal fibrinogen.
In this study we identified the presence of normal
fibrinogen in patients who had AL with prolonged thrombin
times and demonstrated the normal action of thrombin on
this isolated fibrinogen. An inhibitor or inhibitors must exist
in the plasma of patients with primary systemic AL,.These
inhibitors are separated by the fibrinogen isolation procedure.
The prolonged thrombin time in AL is not due to
dysfibrinogenemia but to an inhibitory activity that remains
in the supernatant after fibrinogen is precipitated.
REFERENCES
1. Camoriano JK, Greipp PR, Bayer GK, Bowie EJW: Resolution of acquired factor X deficiency and amyloidosis with melphaIan and prednisone therapy. N Engl J Med 316:1133,1987
2. Butler WM, Baldwin PE: Prolongation of thrombin and
reptilase times in patients with amyloidosis and acquired factor X
deficiency. South Med J 77:648,1984
3. McPherson RA, Onstad JW,
Ugoretz RJ, Wolf P L Coagulopathy in amyloidosis: Combined deficiency of factors IX and X. Am
J Hematol3:225, 1977
4. Galbraith PA, Sharma N, Parker WL, Kilgour JM: Acquired
factor X deficiency: Altered plasma antithrombin activity and
association with amyloidosis. JAMA 230:1658, 1974
5. Khoory MS, Nesheim ME, Bowie EJW, Mann KG: Circulating heparan sulfate proteoglycan anticoagulant from a patient with
a plasma cell disorder. J Clin Invest 65:666,1980
6. Soria J, Soria C, Ryckewaert JJ, Samama M, Thomson JM,
Poller L Study of acquired dysfibrinogenemia in liver disease.
Thromb Res 19:29,1980
7. Palascak JE, Martinez J: Dysfibrinogenemia associated with
liver disease. J Clin Invest 60:89,1977
8. Bowie EJW, Thompson JH Jr, Didisheim P, Owen CA Jr:
Mayo Clinic Laboratory Manual of Hemostasis. Philadelphia, PA,
Saunders, 1971, p 97
9. Straughn W 111, Wagner RH: A simple method for preparing
fibrinogen. Thromb Diath Haemorrh 16:198,1966
10. Morrison PR: Preparation and properties of serum and
plasma proteins. XV. Some factors influencing the quantitative
determination of fibrinogen. J Am Chem SOC69:2723,1947
11. Jim RTS: A study of the plasma thrombin time. J Lab Clin
Med 50:45,1957
12. Soulier J-P: A new adsorption agent for coagulation factors.
J Clin Pathol 12:303,1959
13. Lucas FV, Fishleder AJ, Becker RC, Cavalier DS, Tubbs
RR: Acquired factor X deficiency in systemic amyloidosis. Cleve
Clin J Med 54:399,1987
14. Yood RA, Skinner M, Rubinow A, Talarico L, &hen AS:
Bleeding manifestations in 100 patients with amyloidosis. JAMA
249:1322,1983
15. Schroeder FM, Miller FJ Jr, Nelson JA, Rankin RS: Gastrointestinal angiographic findings in systemic amyloidosis. AJR
Am J Roentgen01 131:143,1978
16. Furie B, Greene E, Furie BC: Syndrome of acquired factor
X deficiency and systemic amyloidosis in vivo studies of the
metabolic fate of factor X. N Engl J Med 297:81, 1977
17. Greipp PR, Kyle RA, Bowie E J W Factor X deficiency in
amyloidosis: A critical review. Am J Hematol 11:443,1981
18. Francis JL, Armstrong DJ: Acquired dysfibrinogenaemia.
Med Lab Sci 40:165,1983
19. Tefferi A, Nichols WL, Bowie EJW: Circulating heparin-like
anticoagulants: Report of five consecutive cases and a review. A m J
Med 88:184,1990
20. Bowman HS: Acquired fibrinogenemia. Am J Med 24:967,
1958
21. Gastineau DA, Kazmier FJ, Nichols WL, Bowie EJW:
Lupus anticoagulant: An analysis of the clinical and laboratory
features of 219 cases. Am J Hematol19:265,1985
From www.bloodjournal.org by guest on October 12, 2016. For personal use only.
1991 77: 2637-2640
Inhibitor of the thrombin time in systemic amyloidosis: a common
coagulation abnormality
DA Gastineau, MA Gertz, TM Daniels, RA Kyle and EJ Bowie
Updated information and services can be found at:
http://www.bloodjournal.org/content/77/12/2637.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.