Administrative Office
St. Joseph's Hospital Site, L301-10
50 Charlton Avenue East
HAMILTON, Ontario, CANADA L8N 4A6
PHONE: (905) 521-6141
FAX: (905) 521-6142
http://www.fhs.mcmaster.ca/hrlmp/
Issue No. 39
QUARTERLY NEWSLETTER
Spring, 1995
ACTIVATED PROTEIN C RESISTANCE AND
THROMBOSIS
In the last two years, a new coagulation disorder, termed "activated protein C resistance" or "APC
resistance" has been identified as an important cause of inherited thrombotic disease. Most individuals with
APC resistance have been shown to have a mutation in the factor V gene that causes the coagulation
factor V protein to escape regulation by the protein C anticoagulant pathway. This results in a disturbance in
the normal balance between hemostasis and clot promotion. Because the mutation associated with this
abnormality occurs in many parts of the world, APC resistance has become recognized as one of the most
frequent causes of inherited, prothrombotic disease.
In health, clot formation is balanced by a natural anticoagulant system, the protein C pathway. The
coagulation factors Va and VIIIa are key non-enzymatic components of the hemostatic pathway. Together,
they accelerate coagulation 300,000 fold by promoting the assembly of procoagulant enzyme complexes.
When coagulation occurs, protein C is activated, and, together with protein S, it inactivates factors Va and
VIIIa by proteolytic cleavage. Although defects in the protein C anticoagulant pathway, due to deficiencies in
protein C or protein S, have been recognized for many years; these disorders occur infrequently in patients
with inherited thrombotic disease. Similarly, defects in antithrombin, another important natural anticoagulant,
are also rare. Together, protein C, protein S and antithrombin deficiencies are found in only 10-15% of
patients with thrombophilia or inherited thrombotic disease.
How was this defect discovered? Studies of a family with inherited thrombosis by Bjorn Dalhback in
Sweden, led to the discovery of an abnormality in factor Va that causes it to escape the normal control by
the protein C pathway. Coagulation, in individuals with this defect, is resistant to the anticoagulant effects of
APC - this discovery led to the term "APC resistance" to describe this condition. A number of laboratories
have confirmed this observation and several researchers have identified the same genetic mutation in many
patients and families with "APC resistance". A single point mutation in the coding region of the factor V gene
is observed in 90% of "APC resistant" individuals. This single, base substitution mutation in the factor V
heavy chain domain changes arg 506 ® gln. This change in the amino acid sequence occurs in the first,
APC cleavage site in factor V. The mutant protein cannot be cleaved by APC at this site, allowing factor Va
to escape downregulation by the protein C pathway. This mutation is common, occurring in approximately
5% of the general population.
What tests are used to detect activated protein C resistance? There are currently two types of tests
used for the investigation of APC resistance: a functional clotting assay and a DNA test for determining the
common factor V gene mutation. The functional assay is a modification of factor V-dependent clotting tests,
in which the clotting times with and without APC are compared, to determine if there is APC resistance. Most
laboratories used a modified aPTT test, where APC is added to the calcium chloride reagent. The results are
expressed as a ratio:
activated protein C resistance ratio = baseline aPTT
(APCR) aPTT with APC added
The degree of functional impairment may be important in determining an individual's risk for thrombosis. A
homozygous individual has a lower ratio than a heterozygote. The aPTT reagent used for the test is an
important determinant of the ratio obtained and the functional assays differ in their ability to discriminate
between normal and abnormal results. Cutoff ratios vary with the reagents used. DNA analysis, looking for
the common mutation in the factor V gene associated with APC resistance, is used for confirmation. DNA
testing is done by PCR amplification of the region of the factor V gene containing the mutation site, followed
by restriction enzyme digestion or dot-blot hybridization to allow detection of the point mutation.
A number of investigators have observed families with inherited APC resistance that lack the arg 506
mutation, suggesting that other genetic causes of APC resistance exist. These may account for as many as
10% of the cases of APC resistance. To date, no other mutations have been identified.
What is the significance of activated protein C resistance? Clinical information regarding the
significance of APC resistance is still emerging. APC resistance may occur in isolation or may be seen with
other inherited prothrombotic states (e.g. protein C deficiency). Investigations suggest that the relative risk
for developing thrombosis with APC resistance may be approximately 5-10 times greater than the normal
population. However, unlike other inherited prothrombotic conditions, homozygous APC resistance is not as
devastating a condition and as many as 50% of individuals homozygous for APC resistance may never
develop thrombosis. The risk of developing thrombosis is lower in individuals who are heterozygous for the
mutation. In patients who receive prophylactic anticoagulant therapy to prevent thrombosis during periods of
high risk, it is not yet known if APC resistance contributes to thrombosis. Until more is known about the
disorder, there is no evidence to indicate that general screening for APC resistance is of value. Because of
the frequency of APC resistance in the population (approximately 5% in our region) it is now the most
commonly identified laboratory abnormality in patients with thrombophilia. Approximately 50% of the patients
we currently investigate for inherited prothrombotic states have APC resistance.
Who should be tested for activated protein C resistance? Testing for APC resistance, along with the
testing for protein C, protein S and antithrombin deficiencies, should be considered in individuals with
idiopathic thrombosis, thrombosis at a young age, or if there is a family history of thrombotic disease. At
present, there is no clinical evidence to support APC resistance testing in other situations.
Catherine P.M. Hayward, MD, FRCPC
The APC resistance test, as well as the DNA assay, is available through the Hamilton Laboratory Reference
Centre. Information as to the cost and the samples required can be obtained through the Laboratory
Reference Centre.
We are looking forward to providing further improvements in service, and welcome comments and enquiries.
Feel free to phone us at 521-5084, or 521-2100, extension 3710.
*********************
The Hamilton Health Sciences Laboratory Program is a collaborative program of Hamilton Civic, St.
Joseph's and Chedoke-McMaster Hospitals, which operate Community Laboratory Services as a service to
Hamilton Physicians and their patients.
For further information concerning the Laboratory Program call Mr. A.J. Bailey at 572-7575 and for
Community Laboratory Services telephone Mrs. K. Williams at 521-6052.
COMMUNITY LABORATORY SERVICES
Collection Centres
Ancaster Centre
226 Wilson Street E.
Ancaster, Ontario
Tel: (905) 648-4080
Charlton Centre
25 Charlton Ave. E.
Hamilton, Ontario
Tel: (905) 521-6052
George St. Centre
196 George St.
Hamilton, Ontario
Tel: (905) 525-1562
Carlisle Centre
1493 Centre Rd.
Carlisle, Ontario
Tel: (905) 689-0818
Dundas Centre
16 Cross St.
Dundas, Ont.
Tel: (905) 627-3814 or
(905) 627-7190
Concession Centre
688 Concession St.
Hamilton, Ontario
Tel: (905) 383-2021
Fennell Centre
836 Fennell Street E
Hamilton, Ontario
Tel: (905) 383-0505 or
(905) 383-9953
East Hamilton Centre
1463 Main St. East
Hamilton, Ontario
Tel: (905) 549-5004
First Place Centre
350 King St. E., Ste. 103
Hamilton, Ontario
Tel: (905) 522-8765/66
North Hamilton Centre
554 John St. North
Hamilton, Ontario
Tel: (905) 522-4197
Stoney Creek Centre
2757 King Street E.
Hamilton, Ontario
Tel: (905) 573-4824
For Housecalls
throughout the region,
telephone Mrs. K.
Williams at (905) 5216052
For Laboratory Reference Centre Services phone Mrs. B. Baltzer at (905) 521-6065 or fax requests for
information to (905) 528-1464