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(Stroke. 1996;27:1163-1166.)
© 1996 American Heart Association, Inc.

Articles

Activated Protein C Resistance in Ischemic Stroke Not Due to Factor V Arginine⁵⁰⁶Glutamine Mutation

Mark Fisher, MD; Jose A. Fernandez, MD, PhD; Sebastian F. Ameriso, MD; Dangci Xie, MD, PhD; Andras Gruber, MD; Annlia Paganini-Hill, PhD John H. Griffin, PhD

the Departments of Neurology (M.F., S.F.A., D.X.) and Preventive Medicine (A.P.-H.), University of Southern California School of Medicine (Los Angeles), and the Departments of Molecular and Experimental Medicine and of Vascular Biology, The Scripps Research Institute, La Jolla, Calif (J.A.F., A.G., J.H.G.).

Correspondence to Mark Fisher, MD, Department of Neurology, University of Southern California School of Medicine, 1333 San Pablo St, MCH 246, Los Angeles, CA 90033. E-mail mjfisher@hsc.usc.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose Resistance to activated protein C (APC), a natural plasma anticoagulant, is the most common identifiable risk factor for venous thromboembolic disease. One point mutation in coagulation factor V that renders it APC-resistant is found in >90% of APC-resistant venous thrombosis patients. To determine the prevalence of APC resistance and of this factor V mutation in stroke, we screened a group of ischemic stroke patients.

Methods Hispanic ischemic stroke patients were screened using two different activated partial thromboplastin time-based assays. One assay using neat patient plasma determined APC resistance, and the other assay using patient plasma diluted into factor V-deficient plasma determined APC-resistant factor V, including the Arg⁵⁰⁶Gln mutation. Results were compared with those in 31 Hispanic control subjects of similar ages.

Results Six of 63 (9.5%) stroke patients had APC resistance compared with none of 31 (0%) control subjects. No patient or control subject had APC-resistant factor V, ie, the factor V Arg⁵⁰⁶Gln mutation.

Conclusions In Hispanic patients with ischemic stroke, the incidence (approximately 10%) of APC resistance is not caused by the factor V Arg⁵⁰⁶Gln mutation. APC resistance not caused by this factor V mutation may be a risk factor for ischemic stroke in this population.

Key Words: cerebral ischemia • Hispanic Americans • protein C • thrombosis

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Activated protein C is a naturally circulating anticoagulant protein in plasma¹ that inhibits activated coagulation factors V and VIII.² Resistance to APC³ is the most frequent identifiable risk factor for VTE and is found in 20% to 60% of white patients with VTE, depending on cohort selection.^{4 5 6} Remarkably, 60% of pregnancy-associated and 30% of oral contraceptive-associated VTE is associated with APC resistance.⁷ Resistance to APC is defined as an abnormally low anticoagulant response to APC. One point mutation in the coagulation factor V gene (GA at nucleotide 1691) that causes replacement of amino acid residue arginine 506 by glutamine is found in approximately 90% of white patients with VTE and APC resistance.^{8 9 10 11} The variant Gln⁵⁰⁶-factor Va molecule is APC resistant^{8 12 13 14} and is inactivated by APC approximately 10 times slower than normal Arg⁵⁰⁶-factor Va,¹⁴ consistent with the hypothesis that this mutation may contribute to a mild prethrombotic state.

Although APC resistance due to the factor V mutation is important in the pathophysiology of VTE, the contribution of APC resistance to ischemic stroke is less clear. One Austrian study indicated that 6 of 30 (20%) stroke patients had APC resistance, on the basis of coagulation assays.¹⁵ Another study of three Italian families suggested a possible relationship between ischemic stroke and APC resistance due to the factor V mutation.¹⁶ However, five DNA-based studies of stroke patients found no increased prevalence of the factor V mutation compared with control groups.^{17 18 19 20 21} The purposes of this study were, first, to address these potentially conflicting results and, second, to consider the utility in the evaluation of ischemic stroke patients of two different APC resistance clotting assays: (1) a "classic" aPTT assay using neat patient plasma and (2) a second-generation aPTT assay using patient plasma diluted into factor V-deficient substrate plasma that is specific for APC-resistant factor V.^{12 22}

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Because the racial and ethnic makeup of the majority of stroke patients at the Los Angeles County-University of Southern California Medical Center is Hispanic and because the factor V Arg⁵⁰⁶Gln mutation shows markedly different prevalences in control populations of different racial and ethnic backgrounds,^{23 24} we studied only Hispanic subjects. Patients with ischemic stroke were participants in ongoing studies of stroke epidemiology and prevention^{25 26} at the University of Southern California School of Medicine. The control group consisted of Hispanic subjects without history of stroke and of comparable age to the stroke patients. Hispanic patients and control subjects in southern California are drawn mainly from the genetic pool of Mexico and Central America. After venipuncture, citrated plasma samples were prepared and stored frozen before assays.

Coagulation-based APC resistance tests were performed on all samples using two different aPTT assays. The first assay, designated assay A, used a slightly modified⁴ version of the classic assay for APC resistance.³ The second assay, assay B, involved a specific test for APC-resistant factor V using factor V-deficient plasma.^{12 22} For assay B, 50 µL of patient plasma diluted 1:10 with factor V-deficient plasma (George King Bio-Medical Inc) was incubated with 50 µL of the aPTT reagent Platelin LS (Organon Teknika) for 5 minutes at 37°C. Then recalcification was performed using 50 µL of 30-mmol/L CaCl₂ solution in Tris-buffered saline, pH 7.4, containing bovine serum albumin either with or without 16 nmol/L human APC. Clotting times were determined with an ST4 coagulometer (American Bioproducts Co). The APC resistance ratio for both assays was calculated as the ratio of clotting time with APC to the clotting time without APC. As normal and abnormal controls, pooled normal plasma and plasma containing APC-resistant factor V from a patient previously identified⁹ as heterozygous for the factor V Arg⁵⁰⁶Gln mutation were used. In the assay B protocol, all of the factor V in the clotting mixture comes from the test plasma, whereas approximately 90% of all other clotting factors comes from the factor V-deficient reagent plasma; thus, the APC resistance of the patient's factor V is directly detected in this assay.^{12 22} In a previous study to validate assay B, 94 consecutive blood samples submitted to the Scripps Clinic Coagulation Laboratory for monitoring oral anticoagulant therapy were assayed using assay B and genotyped for the factor V mutation of Arg⁵⁰⁶Gln; when the clotting assay results (7 of 94 abnormals) were compared with results for DNA genotyping, both the sensitivity and the specificity of assay B were 100% for the factor V Arg⁵⁰⁶Gln mutation (J.H. Griffin, T. Calkins, D. Byland, unpublished data, 1995). Mean and SD values for APC resistance ratios were calculated on data log transformed to achieve normality and then reconverted back to the original scale, as previously described.⁵ Screening for factor V mutation (nucleotide 1691 GA) was performed as described,^{9 27} using DNA extracted from whole blood (Qiagen Inc). Unpaired Student's t tests were used to test for differences between groups; P<.05 was considered statistically significant. The study was approved by the University of Southern California Institutional Review Board.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Plasma samples from 63 Hispanic patients with ischemic stroke and from 31 Hispanic control subjects of similar age were tested for APC resistance using assay A and for APC-resistant factor V using assay B. Clinical descriptions of the subjects are summarized in the Table. Time from stroke to venipuncture was less than 4 days for 36 patients; for 27 patients, the time from stroke to venipuncture ranged from 1 month to more than 3 years. APC resistance ratios did not differ significantly between male and female control subjects (P>.40 and P>.35 for assays A and B, respectively) or between stroke patients studied less than 4 days after the stroke and those studied later (P>.15 and P>.40 for assays A and B, respectively).

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Study Subjects

Data for APC resistance from assay A are shown in the Figure, left. For the control group, the mean value of the APC resistance ratio in assay A was 3.40, and the mean±2 SD ranged from 2.29 to 5.06. The mean value for the 63 patients was 3.10 (Figure, left). None of the 31 control subjects but 6 of 63 (9.5%) patients were APC resistant, ie, below the 2.29 cutoff value (2 SDs below the mean in the control subjects).

View larger version (29K): [in this window] [in a new window]   Figure 1. APC resistance and APC-resistant factor V in ischemic stroke patients. Left, APC resistance of 63 ischemic stroke patients and 31 control subjects as determined with assay A, a "classic" aPTT-based test using neat subject plasma (see "Subjects and Methods"). The arrows indicate the mean values for patients and control subjects (3.10 and 3.40, respectively). The solid lines indicate the cutoff values (mean±2 SD) for the control panel. Values below 2.29 were abnormal. The value of the APC resistance ratio for pooled normal plasma was 3.06, and for plasma from a control subject who is heterozygous for the factor V Arg506Gln mutation, the value was 1.60 (data not shown). Not shown are points for values from two patients and one control subject that were >6. Right, Plasma APC-resistant factor V of 63 ischemic stroke patients and 31 control subjects. APC-resistant factor V was determined on the basis of APC resistance ratios using assay B, an aPTT-based test using 1:10 dilutions of subject plasma in factor V-deficient substrate plasma (see "Subjects and Methods"). The arrows indicate the mean values for patients and control subjects (2.87 and 2.78, respectively). The solid lines indicate the cutoff values (mean±2 SD) for the control panel. Values below 1.78 were abnormal. The value of the APC resistance ratio for pooled normal plasma was 2.70, and for plasma from a subject who is heterozygous for the factor V Arg506Gln mutation, the value was 1.50 (data not shown). Not shown is one point for one control value that was >5.

Data from assay B for APC-resistant factor V are shown in the Figure, right. For the control group, the mean value of the APC resistance ratio in assay B was 2.78, and the mean±2 SD ranged from 1.78 to 4.35. The mean value for the 63 patients was 2.87 (Figure, right). No control subject or patient had a value below the 1.78 cutoff value (2 SDs) for APC-resistant factor V.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Using assay A to determine APC resistance of plasma samples, we found APC resistance in 9.5% (6/63) of Hispanic ischemic stroke patients versus 0% (0/31) in a group of similarly aged Hispanic control subjects. These positive findings are consistent with the observation that 20% of Austrian stroke patients were APC resistant when a similar clotting assay was used to define APC resistance.¹⁵ The Austrian study included 30 patients (18 females and 12 males) with a mean age of 46.6 years; 3 males and 3 females were APC resistant. In our Hispanic patient group of 63 patients (29 females and 34 males), with a mean age of 57.1 years, 3 females and 3 males were APC resistant.

Using assay B to detect abnormal factor V that is APC resistant, we found no Hispanic stroke patient or control subject to show positive results for the presence of APC-resistant factor V. This finding is consistent with recent DNA-based studies^{17 18 19 20 21} reporting no increased prevalence of the factor V Arg⁵⁰⁶Gln mutation in stroke patients. These findings suggest that the increased prevalence of APC resistance in stroke patients is not caused by APC-resistant factor V molecules, such as Gln⁵⁰⁶-factor V.

Since APC resistance in approximately 90% of VTE cases is explained by the factor V Arg⁵⁰⁶Gln muta-tion,^{8 9 10 11 24 27 29} it was surprising that none of our 6 stroke patients with APC resistance had APC-resistant factor V. Our data emphasize that clotting assays for APC resistance that use neat plasma from stroke patients can detect functional abnormalities of the protein C pathway not due to APC-resistant factor V. In a seminal study of APC resistance, heredity, and VTE, Zoller et al¹¹ reported that although the factor V mutation and APC resistance showed perfect cosegregation in 40 of 50 Swedish families, the factor V mutation was not found in three APC-resistant families. Furthermore, in seven other families, 12 of 57 APC-resistant individuals lacked the factor V mutation; there was a significant difference in the thrombosis-free survival between those with and those without APC resistance. Thus, factor V-independent APC resistance appears to be a risk factor for VTE, and other as yet unknown genetic defects may cause APC resistance.¹¹ There is currently no information indicating whether the factor V-independent APC resistance associated with VTE involves the same molecular defects as are associated with ischemic stroke. Although the molecular defects remain to be identified, our study confirms the results of an Austrian study¹⁵ and suggests that factor V-independent APC resistance may be a risk factor for ischemic stroke in some subjects.

Possible molecular defects that might cause such APC resistance without APC-resistant factor V^{24 28 29} include as yet undescribed APC-resistant factor VIII molecules, elevated plasma levels of factor VIII,³⁰ dysfunctional protein S molecules, various hormone therapies including oral contraceptive use, and lupus anticoagulants and/or antiphospholipid antibodies.³¹ Several of these possible explanations (eg, dysfunctional protein S, oral contraceptive use, or antiphospholipid antibodies) are themselves associated with an increased risk of venous and/or arterial thrombosis.^{28 29 31 32} The mechanism(s) of APC resistance in our stroke patients is currently under investigation; further research is needed to identify molecular defects responsible for factor V-independent APC resistance in various thrombosis patient groups and to demonstrate whether these defects contribute to the pathophysiology of venous or arterial thrombosis. Our current working hypothesis is that APC resistance involves one or more abnormalities that increase risk for ischemic stroke.

On the basis of available information, we recommend against the use of DNA-based assays or clotting assays specific for the factor V Arg⁵⁰⁶Gln mutation for the routine evaluation of adult stroke patients. Nonetheless, subgroups of stroke patients in whom investigations for APC resistance are currently warranted include (1) Hispanics with ischemic stroke, (2) stroke patients with a history of VTE,^{3 4 5 6 8 9 10 11} (3) patients with intracranial venous sinus thrombosis,³³ (4) patients with stroke associated with the use of oral contraceptives,³² (5) migraine patients with stroke,¹⁸ and (6) stroke patients with antiphospholipid antibody syndrome.³¹

We studied Hispanic subjects, most of whom came to southern California from Mexico and Central America. Hispanics tend to have a lower stroke rate compared with whites; this has been attributed to a lower prevalence of hypertension among Hispanics.³⁴ No APC-resistant factor V was found in 94 subjects (63 stroke patients plus 31 control subjects). This suggests that Arg⁵⁰⁶Gln mutation in factor V is not a polymorphism in Hispanics; however, a larger sample size will be needed to confirm this finding. Although the allelic frequency of the factor V Arg⁵⁰⁶Gln mutation in European and American whites ranges from 1% to 6% (mean, 2.4%),^{23 24 29} this allele is nearly absent in other populations (eg, Asians who live in Asia, blacks who live in Africa, and perhaps Indians native to North and South America).²³ We do not yet know whether our findings are specific for the Hispanic population. Nevertheless, these data emphasize the importance of choosing appropriate controls for comparison with patients in clinical studies of APC resistance and the necessity for caution in extrapolation of data from one racial and ethnic group to another.

The role of APC in stroke pathogenesis has received limited attention. However, several reports point to the potential importance of this circulating anticoagulant. Levels of APC are decreased in acute ischemic stroke.³⁵ Moreover, thrombomodulin (the endothelial cofactor for protein C activation by thrombin) has limited expression in the human brain,³⁶ particularly in brain regions where infarction is common.³⁷ The modest frequency (9.5% to 20%) of APC resistance in brain infarction does not contradict the hypothesis that local (ie, CNS) production of APC may be a limiting factor and have a significant role in ischemic stroke mechanisms. Ischemic insult induces rapid activation of protein C in the porcine coronary microcirculation, and blocking this protein C activation significantly impairs recovery from ischemia.³⁸ Protein S, an anticoagulant cofactor for APC, is synthesized by CNS-derived cells in culture and perhaps in the CNS in vivo, where it may contribute antithrombotic activity.³⁹ Thus, we speculate that the protein C pathway may exert important antithrombotic activities in the brain and that its impairment, as in the case of any APC resistance, may contribute to stroke.

In conclusion, we report that factor V-independent APC resistance is present in approximately 10% of Hispanic patients with ischemic stroke syndromes, whereas APC-resistant factor V and specifically the factor V Arg⁵⁰⁶Gln mutation are absent. Screening for APC resistance is not yet routinely indicated for all stroke patients but is an important topic for research. However, assessments for APC resistance are warranted in Hispanic stroke patients and in non-Hispanic stroke patients with VTE, intracranial venous sinus thrombosis, oral contraceptive usage, migraine, or antiphospholipid antibody syndromes.

	Selected Abbreviations and Acronyms

 

APC = activated protein C aPTT = activated partial thromboplastin time CNS = central nervous system VTE = venous thromboembolic disease

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants P01NS31945 and NS24456 (Dr Fisher) and R37HL52246 (Dr Griffin), by the Stein Endowment Fund (Dr Griffin), and by a grant from Sanofi-Winthrop (Dr Fisher). We gratefully acknowledge the assistance of Edward Lozano, Aldana Martin, Patricia Moran, Pey-Jiuan Lee, Hamid Rabiee, Soma Sahai, and Andrea Scicli. We thank Xiao Xu for the DNA analysis of the factor V mutation.

Received January 30, 1996; revision received March 29, 1996; accepted April 1, 1996.

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				M. C.H. de Visser, F. R. Rosendaal, and R. M. Bertina A Reduced Sensitivity for Activated Protein C in the Absence of Factor V Leiden Increases the Risk of Venous Thrombosis Blood, February 15, 1999; 93(4): 1271 - 1276. [Abstract] [Full Text] [PDF]

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				J. F. Meschia, J. Biller, T. Witt, A. Greist, and S. N. Rhinehart Is Hormone Replacement a Risk Factor for Ischemic Stroke in Women With Factor V Leiden Mutation? Arch Neurol, August 1, 1998; 55(8): 1137 - 1139. [Abstract] [Full Text] [PDF]

				W. T. Longstreth Jr, F. R. Rosendaal, D. S. Siscovick, H. L. Vos, S. M. Schwartz, B. M. Psaty, T. E. Raghunathan, T. D. Koepsell, and P. H. Reitsma Risk of Stroke in Young Women and Two Prothrombotic Mutations: Factor V Leiden and Prothrombin Gene Variant (G20210A) Stroke, March 1, 1998; 29(3): 577 - 580. [Abstract] [Full Text] [PDF]

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				A. Zivelin, J. H. Griffin, X. Xu, I. Pabinger, M. Samama, J. Conard, B. Brenner, A. Eldor, and U. Seligsohn A Single Genetic Origin for a Common Caucasian Risk Factor for Venous Thrombosis Blood, January 15, 1997; 89(2): 397 - 402. [Abstract] [Full Text] [PDF]

				R. L. Brey and B. M. Coull Cerebral Venous Thrombosis: Role of Activated Protein C Resistance and Factor V Gene Mutation Stroke, October 1, 1996; 27(10): 1719 - 1720. [Full Text]

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