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Activated Protein C Resistance in Ischemic Stroke Not Due to Factor V Arginine506->Glutamine Mutation
Abstract
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.
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 Arg506-->Gln mutation. Results were compared with those in 31 Hispanic control subjects of similar ages.
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 Arg506-->Gln mutation.
In Hispanic patients with ischemic stroke, the incidence (approximately 10%) of APC resistance is not caused by the factor V Arg506-->Gln mutation. APC resistance not caused by this factor V mutation may be a risk factor for ischemic stroke in this population.
Supplementary resource (1)
Data
January 2014
... aPCR was originally discovered in thrombophilic families and later shown to be associated with the common FV p.506R [ Q (FV Leiden) mutation, which abolishes one of the aPC-cleavage sites in FV [22]. Although FV Leiden is the major cause of hereditary aPCR, it is becoming increasingly clear that several other genetic and acquired conditions contribute to aPCR and thereby increase the risk of venous thrombosis [18][19][20][21]. The factor V Cambridge, Hong Kong and Liverpool mutations can also lead into the aPCR phenotype [2,18], as can the presence of LA and/or antiphospholipid antibodies [18,19], increased factor VIII levels [18,20], dysfunctional protein S molecules, various hormone therapies including oral contraceptive use [21], cancer [20] and the use of DOACs [22][23][24]. ...
... Although FV Leiden is the major cause of hereditary aPCR, it is becoming increasingly clear that several other genetic and acquired conditions contribute to aPCR and thereby increase the risk of venous thrombosis [18][19][20][21]. The factor V Cambridge, Hong Kong and Liverpool mutations can also lead into the aPCR phenotype [2,18], as can the presence of LA and/or antiphospholipid antibodies [18,19], increased factor VIII levels [18,20], dysfunctional protein S molecules, various hormone therapies including oral contraceptive use [21], cancer [20] and the use of DOACs [22][23][24]. The C4.5 machine learning algorithm did not reveal any hidden associations between the case attributes but rather expected ones: FV Leiden mutation predicted aPCR phenotype which by itself predicted the use of DOACs. ...
Activated protein C resistance (aPCR) phenotypes represent around 20% of the laboratory findings in Mexican Mestizos having suffered thrombosis and displaying clinical markers of thrombophilia. In a single institution for a 276-month period, 96 Mexican mestizos with a history of thrombosis and clinical markers of a primary thrombophilic state were prospectively studied to identify a thrombophilic condition. An abnormal aPCR phenotype was identified in 18 individuals. Evaluation of those with an abnormal aPCR phenotype, identified that 44% had factor V Leiden mutation, 22% increased levels of factor VIII, 16% anti-phospholipid antibodies and 6% a lupus anticoagulant. In the remaining 22%, the use of direct oral anticoagulants (DOACs) in the past period of 12–24 h was recorded. We found significant associations between abnormal aPCR phenotype and the factor V Leiden mutation (p = 0022), between abnormal aPCR phenotype and the use of DOACs (p = 0.006) and between antiphospholipid antibodies and lupus anticoagulant (p < 0.0001). These data are consonant with those observed in other populations and further identify that consideration be given to identifying whether individuals are being treated with the novel DOACs when conducting laboratory studies oriented to identify the etiology of thrombosis.
... Se sabe que en el 90% de los casos de resistencia a la proteína C activada (RPCA), el mecanismo de este trastorno de la coagulación es la presencia de factor Leiden V [25]. Fisher et al [27], sin embargo, encontraron RPCA sin factor V Leiden en 6 de 63 (9,5%) pacientes con ictus isquémico y en ninguno de los 31 (0%) sujetos controles. Como veremos a continuación, el tamaño de la muestra parece insuficiente para inferir de forma definitiva que la RPCA no ligada a mutaciones conocidas del gen del factor V sea un factor de riesgo de ictus isquémico. ...
The aetiology, investigation and outcome of ischaemic stroke were studied in a population of 128 children. Cerebrovascular abnormalities were present in the majority of children; in many cases these conformed to specific diagnostic categories, with implications for management. In contrast, previously unrecognised non-vascular risk factors for stroke were relatively rare. In particular, the prevalence of inherited prothrombotic states was no higher in children with stroke than in control populations. Although magnetic resonance angiography was useful in identifying cerebrovascular lesions, conventional cerebral angiography had a continuing and definable role in the investigation of the child with ischaemic stroke. In the investigation of outcome after ischaemic stroke a simple questionnaire investigating parents' perception of residual disability was shown to correlate well with therapists' and neuropsychological assessment. Over half the children in this population had significant residual deficits; the incidence of recurrent stroke was 17% over 5 years. A younger age at the time of stroke was associated with worse outcome. However, prognosis was not influenced by other clinical factors. In a subgroup of 38 children with lesions in the territory of a middle cerebral artery, although the location of the lesion was not related to outcome, outcome was poor in all patients who had infarcted at least 10% of intracranial volume had. Lesion size could, therefore, be used to identify patients at high risk of long term disability for future treatment trials. These findings support the view that there is a role for both acute treatment and secondary prevention in children with ischaemic stroke. This study has characterised in detail a large population of children with ischaemic stroke and has given rise to several practical recommendations about the investigation and management of such patients.
Objective.—To review the current state of the art regarding the role of the clinical laboratory in diagnostic testing for the factor V Leiden (FVL) thrombophilic mutation (and other protein C resistance disorders), and to generate, through literature reviews and opinions of recognized thought-leaders, expert consensus recommendations on methodology and diagnostic, prognostic, and management issues pertaining to clinical FVL testing.
Data Sources, Extraction, and Synthesis.—An initial thorough review of the medical literature and of current best clinical practices by a panel of 4 experts followed by a consensus conference review, editing, and ultimate approval by the majority of a panel of 28 additional coagulation laboratory experts.
Conclusions.—Consensus recommendations were generated for topics of direct clinical relevance, including (1) defining those patients (and family members) who should (and should not) be tested for FVL; (2) defining the preferred FVL laboratory testing methods; and (3) defining the therapeutic, prophylactic, and management ramifications of FVL testing in affected individuals and their family members. As FVL is currently the most common recognized familial thrombophilia, it is hoped that these recommendations will assist laboratorians and clinicians caring for patients (and families) with this common mutation.
Arterial thrombosis (AT) is a pivotal event in the natural history of coronary heart disease (CHD), stroke (cerebrovascular ischemic disease), and peripheral vascular disease. CHD represents a continuum of myocardial ischemia ranging from unstable angina to coronary artery disease (CAD), and to myocardial infarction (MI). Although a family history of CAD is a risk factor for cardiovascular ischemic episodes, the overall impact of family history is modest. Common forms of AT segregate in a non-Mendelian inheritance pattern. Over 95% of early CHD cases are not caused by monogenic defects such as familial hypercholesterolemia (FH), but rather by multiple genetic and environmental determinants. The pathogenesis of AT is probably different from that of venous thrombosis (VT). Most forms of AT are multifactorial disorders resulted from many different combinations of genetic prothrombotic polymorphisms and environmental atherogenic factors, even though some by themselves may not be sufficient to cause disease. Environmental risk factors for AT include high fat diets, cigarette smoking, increased salt consumption, and decreased intake of folate.
Activated protein C (APC) resistance caused by the factor V Leiden mutation is associated with an increased risk of venous thrombosis. We investigated whether a reduced response to APC, not due to the factor V point mutation, is also a risk factor for venous thrombosis. For this analysis, we used the Leiden Thrombophilia Study (LETS), a case-control study for venous thrombosis including 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. All carriers of the factor V Leiden mutation were excluded. A dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis: the lower the normalized APC sensitivity ratio, the higher the associated risk. The risk for the lowest quartile of normalized APC-SR (<0.92), which included 16.5% of the healthy controls, compared with the highest quartile (normalized APC-SR > 1.05) was greater than fourfold increased (OR = 4.4; 95% confidence interval, 2.9 to 6.6). We adjusted for VIII:C levels, which appeared to affect our APC resistance test. The adjusted (age, sex, FVIII:C) odds ratio for the lowest quartile was 2.5 (95% confidence interval, 1.5 to 4.2). So, after adjustment for factor VIII levels, a reduced response to APC remained a risk factor. Our results show that a reduced sensitivity for APC, not caused by the factor V Leiden mutation, is a risk factor for venous thrombosis.
The protein C system is an important natural anticoagulant pathway. Protein C is the key component of the system and it is activated by thrombin bound to thrombomodulin on the surface of endothelial cells. Activated protein C (APC) inhibits coagulation by cleaving and inactivating coagulation factors factor Va and factor Villa. Until recently, the major genetic causes of familial venous thrombophilia were inherited deficiencies of protein C, protein S or antithrombin, but together they were found in less than 5‐10% of patients with thrombosis. In 1993, the situation changed drastically with the description of inherited APC‐resistance as a novel risk factor for venous thrombosis. APC‐resistance is characterized by a poor anticoagulant response to APC. Inherited APC‐resistance is the most common genetic risk factor for this disease and it is found in 20‐60% of patients. The condition is caused by a single point mutation in the gene for factor V which predicts substitution of arginine (R) at position 506 with a glutamine (Q). Mutated factor V (FVR506Q, FV:Q506 or FV Leiden) expresses normal procoagulant properties but is partially resistant to APC. The resulting hypercoagulable state confers a life‐long increased risk of venous but not arterial thrombosis. The FVR506Q mutation is common in Caucasians with a prevalence of 1‐15%, whereas it is not found in other human races. The FVR506Q mutation may, due to its high prevalence, be an additional risk factor in individuals carrying other inherited defects such as deficiency of protein S, protein C or antithrombin. Such individuals have a high incidence of thrombosis and severe thrombophilia is a multigenetic disease. The high prevalence of inherited APC‐resistance and the availability of easy functional and genetic tests will stimulate the development of prophylactic regimens and hopefully result in a decreased incidence of thrombosis.
Stroke is the third leading cause of death in the western world. Each year, approximately 500,000 people in the United States experience a stroke; for 150,000 of them the outcome is fatal. The US National Health Interview Survey indicates that the prevalence of stroke in the United States is 720/100,000 among the white population and 910/100,000 in the nonwhite population [1].
Several genetic variants are currently identified as risk factors for venous and arterial thrombosis (deep venous thrombosis, myocardial infarction and stroke). Activated protein C resistance due to the factor V Leiden (FVL) and the 20210 G>A mutation in the factor II (FII, Prothrombin) gene are well-established causes of thrombophilia. Concerning the risk of myocardial infarction and stroke the results are different: There are studies reporting positive (Ma et al. 1996; Montaruli et al. 1996; Nabavi et al. 1998; Simioni et al. 1995; Arruda et al. 1997; Doggen et al. 1998; Rosendaal et al. 1997; Watzke et al. 1997; Corral et al. 1997) or neutral influences (Ardissino et al. 1996; Sanchez et al. 1997; Schröder et al. 1999; Corral et al. 1997; Kapur et al. 1997; Eikelboom et al. 1998; Ferraresi et al. 1997; Prohaska et al. 1999). The 677C>T mutation in the methylenetetrahydrofolate reductase gene (677C>T MTHFR), which caused a mild hyperhomocysteinemia is considered as a risk factor for coronary heart disease (Kluijtmans et al. 1996; Morita et al. 1997), venous thrombosis and stroke (Margaglione et al. 1998; Kluijtmans et al. 1999), but the results are controversial.
Purpose: Recently there are numerous reports demonstrating environmental and genetic factors in the pathogenesis of thrombotic events. In this case-control study we investigated the relation between genetic and other risk factors and thrombotic events in patients with venous thromboembolism (VTE), cerebral ischemic infarction (CI) and acute myocardial infarction (AMI). Methods: We have investigated the prevalence of the factor V G1691A (FV Leiden) mutation, prothrombin (PT) G20210A, C677T methylene tetrahydrofolate reductase (MTHFR) gene single nucleotide polymorphism in 35 patients with VTE, 21 cases with CI and 63 patients with AMI. Results: FVL carrier rate was found as 37.1% in VTE, 12.7% in AMI and, 14.3% in CI patients. The frequency of PT G20210A mutation was 8.6% in VTE, 6.3% in AMI and 4.8% in CI patients. FVG1691A and PT G20210A mutations were found as significant risk factors for VTE patients compared to controls (P < 0.001). In the control group consisting 81 healthy subjects, protein C deficiency was demonstrated in 1.2% of the subjects. None of the controls had antithrombin or protein S deficiency whereas carrier rate for FVL was 4.9% with an allele frequency of 3.1%. MTHFR C677T mutation was detected in 43 of the healthy controls and one individual was heterozygous for prothrombin gene mutation. Risk assessment of prothrombotic factors revealed only hyperhomocysteinemia as a risk factor for AMI (P<0.05; OR=7.0, 95% CI: 3.4-565). The contribution of other prothrombotic conditions such as MTHFR mutation, PC, PS and AT deficiencies was not significant for the development of venous or arterial thrombotic disease. Conclusion: Our findings suggest that FVG1691A and PT G20210A mutations are significant genetic risk factors contributing to the pathophysiology of venous thrombosis in young Turkish adult population.
A defect involving poor anticoagulant response to activated protein C (APC), an anticoagulant serine protease known to inactivate factors Va and VIIIa in plasma, was recently reported and the existence of a novel APC cofactor was suggested. To define the frequency of this defect among 25 venous thrombophilic patients with no identifiable laboratory test abnormality and among 22 patients previously identified with heterozygous protein C or protein S deficiency, the APC-induced prolongation of the activated partial thromboplastin time assay for these patients was compared with results for 35 normal subjects. The results show that this new defect in anticoagulant response to APC is surprisingly present in 52% to 64% of the 25 patients, ie, in the majority of previously undiagnosed thrombophilia cases, but is not present in 20 of 22 heterozygous protein C or protein S deficient patients, suggesting that the new factor is a risk factor independent of protein C or protein S deficiency. The results demonstrate that abnormalities in the anticoagulant protein C pathway are present in the majority of thrombophilic patients.
A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg⁵⁰⁶ → Gln). Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor VI, and patient II, factor VII), who are homozygous for this mutation. The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring α-thrombin formation. The cleavage and subsequent inactivation by activated protein C (APC) of the α-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va. In normal factor Va, cleavage at Arg⁵⁰⁶ generates a Mr = 75,000 fragment and a Mr = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg⁵⁰⁶ and Arg⁶⁷⁹. Proteolysis at these sites leads to the appearance of Mr = 45,000 and 30,000 fragments and a Mr = 22,000/20,000 doublet. Cleavage at Arg⁵⁰⁶ is membrane-dependent and is required for complete inactivation. Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor VaI and factor VaII retain approximately 50% of their initial activity. After 1 h of incubation with APC, factor VaI retains 20% of its initial cofactor activity whereas factor VaII has 10% remaining cofactor activity. The initial loss in cofactor activity (∼70%) of membrane-bound factor VaI and factor VaII during the first 10 min of the inactivation reaction is correlated with cleavage at Arg⁵⁰⁶ and appearance of a Mr = 45,000 fragment and a Mr = 62,000/60,000 doublet. Subsequently, the Mr = 62,000/60,000 doublet is cleaved at Arg⁶⁷⁹ to generate a Mr = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor VI and factor VII, were inactivated following cleavage at Arg⁵⁰⁶ and Arg⁶⁷⁹, with APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that: 1) cleavage at Arg⁵⁰⁶ is required for optimum exposure of the cleavage sites at Arg⁵⁰⁶ and Arg⁶⁷⁹ and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg⁵⁰⁶ by APC on membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals. Thus cleavage at Arg⁵⁰⁶ and Arg⁶⁷⁹ and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg⁵⁰⁶ for optimum exposure.
The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma- derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.
A coagulation test abnormality, termed activated protein C (APC) resistance, involving poor anticoagulant response to APC is currently the most common laboratory finding among venous thrombophilic patients. Because the anticoagulant activity of APC involves inactivation of factors Va and VIIIa, studies were made to assess the presence of abnormal factors V or VIII. Diluted aliquots of plasma from two unrelated patients with APC resistance and thrombosis were added to either factor VIII-deficient or factor V-deficient plasma and APC resistance assays were performed. The results suggested that patients' factor V but not factor VIII rendered the substrate plasma APC resistant. When factor V that had been partially purified from normal or APC resistant patients' plasmas using immunoaffinity chromatography was added to factor V-deficient plasma, APC resistance assays showed that patients' factor V or factor Va, but not normal factor V, rendered the substrate plasma resistant to APC. Studies of the inactivation of each partially purified thrombin-activated factor Va by APC suggested that half of the patients' factor Va was resistant to APC. These results support the hypothesis that the APC resistance of some venous thrombophilic plasmas is caused by abnormal factor Va.
Protein C activation by thrombin is significantly accelerated by the endothelial cell cofactor, thrombomodulin. In this study, we have developed a radioimmunoassay for thrombomodulin and have measured the cofactor content in several human tissues. The assay method detects as little as 2 ng of thrombomodulin. The highest thrombomodulin content was found in lung and placenta, but the antigen was also detected in spleen, pancreas, liver, kidney, skin, heart, and aorta. Unexpectedly, thrombomodulin was absent from brain. Extracts from cerebral cortex, cerebellum, centrum semiovale, midbrain, basal ganglia, pons, and medulla were devoid of thrombomodulin. In contrast, thrombomodulin antigen is present in extracerebral intracranial vessels, including basilar and internal carotid arteries and choroid plexus, as well as in endothelium of the pia-arachnoid.
The performances of nine commercial kits and an in-house method (HM) for the quantitation of anticardiolipin antibodies (ACA) have been evaluated in a multicenter study. Ninety control and patient samples and six standards from Louisville University were run with kits and with the HM. Marked differences in positivity rate between kits were observed, ranging from 31 to 60% for IgG and 6 to 50% for IgM. Concordance between kits occurred in 59 and 51% of samples for IgG and IgM respectively. Concordance coefficients (kappa) ranged from 0.13 to 0.92. Slopes of regression lines between the declared units of Louisville standards and the units measured from the calibrators of the kits showed great diversity and ranged from 0.159 to 0.931 for IgG and from 0.236 to 0.836 for IgM. The β2-glycoprotein I (β2-GPI) content of the dilution buffers and the wells supplied with the kits revealed noticeable differences. However samples containing anti-(β2-GPI antibodies were classified similarly by all but one kit. In contrast the ability to measure samples devoid of anti-β2-GPI antibodies differed markedly between the kits.
This study shows that differences in positivity rates between the commercial kits may contribute to the differences in ACA prevalence rate found in the literature. The choice of cut-off levels may partly explain the moderate concordance between the kits. In addition some samples behave very differently depending on the kits. In spite of the expression of results in PL units, standardization of ACA assays has not been achieved.
Thrombomodulin, an integral membrane protein with important anticoagulant properties, was evaluated for its distribution in blood vessels of human brain. Adjacent sections from frozen human brain were stained for von Willebrand Factor (an endothelium marker) or thrombomodulin. The number of thrombomodulin-positive blood vessels was expressed as a percentage of all blood vessels in a specific area. The density ratio was compared among cortical and subcortical regions in the human brain. Our results indicated that thrombomodulin density in neocortex, cerebellum, medulla and hippocampus was similar. There was, however, significantly less thrombomodulin in putamen (P less than 0.01), pons (P less than 0.001) and mesencephalon (P less than 0.001) compared to neocortex. The regional distribution of thrombomodulin may prove to be helpful for understanding the development of thrombotic diseases of the brain.