Ischemic Stroke in Young Patients With Activated Protein C Resistance
A Report of Three Cases Belonging to Three Different Kindreds
Background A new pathological condition termed “activated protein C (APC) resistance” has recently been reported to be the most common hereditary blood coagulation disorder associated with familial thrombosis. APC resistance is characterized by a poor anticoagulant response to APC in the plasma of patients and is due to a defect of factor V.
Case Descriptions This report deals with three Italian families with inherited APC resistance in which stroke had occurred at a young age in one of the family members. One of the patients exhibited ischemic stroke at 8 months of age. Although deep vein thrombosis is considered the main clinical manifestation of the defect, its possible association with stroke is discussed. DNA analysis confirmed the presence of the 1691GA mutation in the factor V gene (factor V Leiden) in all patients with a normalized APC sensitivity ratio of less than 0.70. In three cases the APC sensitivity ratios were very low (approximately 1.2), with a normalized APC sensitivity ratio of approximately 0.4. DNA analysis confirmed that these patients were homozygous for the mutation. The clinical history of these patients suggests that homozygosity for the defect is compatible with life and does not seem to be associated with early or more severe thrombophilia compared with homozygous defects of other clotting inhibitors.
Conclusions The cases reported here suggest a possible association of inherited APC resistance with ischemic stroke in young patients. Case-control studies should be performed to assess the true association.
During the last 10 years it has been demonstrated that the protein C/protein S (PC/PS) system and antithrombin III play a major role in the regulation of hemostasis by acting as physiological inhibitors. In fact, inherited defects of these components have been clearly demonstrated to be associated with thrombophilia. A new pathological condition involving the PC/PS system has recently been reported to also be associated with familial thrombosis.1 It has been suggested that activated protein C (APC) resistance, which is characterized by a poor anticoagulant response to APC in the plasma of patients, is due to a selective defect in an anticoagulant function of factor V.2 The final demonstration of this has been obtained by Bertina and coworkers,3 who proved that a mutation (Arg506→Gln) in blood coagulation factor V was associated with resistance to APC. The defect is inherited as an autosomal dominant trait, and its prevalence in different series of patients with a history of thrombosis has been found to vary between 21% and 64%.4 5 6 We found a 15.2% prevalence of the defect in a series of 151 Italian patients with objectively proven deep vein thrombosis (DVT). Therefore, this hereditary defect of factor V is the most common hereditary blood coagulation disorder predisposing to thrombosis identified thus far. In this report we describe three Italian families in which the phenotype and genotype of inherited APC resistance have been detected. Three points will be discussed: (1) the possible association of the defect with ischemic stroke in juveniles and young adults, (2) the clinical behavior of homozygous and heterozygous patients with respect to thrombotic manifestations, and (3) the interference of this factor V abnormality with PC and PS clotting assays based on the activated partial thromboplastin time (aPTT) method.
The proposita (II-1; see Fig 2⇓) is a 44-year-old woman who at the age of 29 experienced a left leg DVT immediately after resection of a right ovarian cyst. After a car accident at the age of 42, she underwent splenectomy and a plaster leg cast for a right tibial fracture. A few days later, because of swelling and pain of the left calf, a left iliofemoral DVT was diagnosed by echo-Doppler with the use of compression ultrasound and by phlebography. Intravenous heparin and warfarin were administered. The patient was discharged after 5 days with oral anticoagulation. Coagulation screening tests performed before heparin was started included fibrinogen, antithrombin III, PC antigen and chromogenic activity, and free and total PS antigen; they were all within the normal range. After 6 months of oral anticoagulation, the patient had a complete recovery from DVT as judged by compression ultrasound, and treatment was discontinued. Fifteen days later she exhibited right-sided hemiparesis. Cerebral CT scan showed a large hypodense ischemic lesion in the left sylvian area involving the parietal, opercular, and nucleocapsular regions. Transcranial ultrasonography suggested an occlusion at the origin of the left middle cerebral artery, which was confirmed by arteriography. Ultrasonography of the neck arteries was normal. Transthoracic and transesophageal (without contrast media) echocardiography showed no source of emboli or the presence of a patent foramen ovale, which might account for paradoxical embolus. Compression ultrasound failed to show recurrences of DVT in the lower limbs. No associated risk factors for juvenile stroke such as cigarette smoking, dyslipidemia, vascular abnormalities, or vasculitis were present.
A new blood sample for coagulation screening was collected; plasma was separated and stored at −80°C. After 1 month of treatment with subcutaneous heparin (7500 U BID), oral anticoagulation was started and permanently maintained. The family history was negative for venous thrombosis and/or stroke.
The propositus (II-4; see Fig 2⇓), a 68-year-old man, experienced a right leg DVT after surgery for inguinal hernia at the age of 48. He experienced three episodes of myocardial infarction at the age of 60 and another episode of DVT during hepatic disease (multiple hepatic abscesses) at the age of 66. The coagulation study, performed in this patient to assess a possible inherited thrombophilia, was inconclusive since the abnormalities found were related to the liver disease. Since several of his relatives had a history of thrombosis, coagulation study was extended to the family. One sister (II-1) at the age of 50 had an ischemic stroke with right-sided hemiparesis 5 days after cholecystectomy. Cerebral arteriography showed an occlusion of the left middle cerebral artery. Impedance plethysmography showed no evidence of venous thrombosis in the lower limbs. No infections could be identified, and both cardiological evaluation and electrocardiogram were normal. No risk factors for stroke were detected at that time. Transthoracic and transesophageal echocardiograms (without “bubble” or contrast study) performed several years later failed to reveal cardiac abnormalities or patent foramen ovale. Carotid duplex ultrasound scanning was normal. The follow-up of the patient was characterized by a partial recovery of the motor function of right upper and lower limbs.
Another sister of the propositus (II-2) experienced two episodes of DVT, one of which occurred during a pregnancy, and also several episodes of superficial phlebitis of both legs. A third sister (II-3) had DVT of the left leg postpartum and also had some episodes of superficial phlebitis. The daughter (III-1) and the son (III-2) of the propositus experienced superficial phlebitis, the former in a leg postpartum and the latter in both arms due to intravenous devices during the postsurgical period after appendectomy.
Complete coagulation screening and DNA analysis were performed in all available family members.
The propositus is an 8-month-old boy (III-1; see Fig 2⇓) admitted to the Pediatric Department of Padua University because of sudden onset of right hemiparesis. A large right parietal hypodense area was documented by CT scan and confirmed 2 days later by MRI. MR angiography (MRA) was performed, and a possible stenosis of the M1 portion of the right middle cerebral artery was documented. A few days after admission the child became tetraplegic. Cerebral CT scan and MRI showed a new left parietal ischemic area (Fig 1⇓). There was no evidence of cerebral venous thrombosis. Homocystinemia level, inflammatory parameters, and complement components were normal. Autoantibodies were not present. Vascular malformation and cardiac abnormalities were excluded by echocardiography (both transthoracic and transesophageal with contrast study) and duplex ultrasound scanning of the extracranial carotid artery. Because of the patient’s clinical condition, carotid angiography was not performed, but MRA imaging seemed to exclude moyamoya disease. No predisposing conditions could be found, and the child had been in previous good health. Prophylaxis with aspirin was given, but 1 month later he developed a massive inferior vena cava thrombosis, which was treated with urokinase (45 000 U bolus, then infusion of 4000 U/kg per hour for 7 days), followed by heparin (1650 U/d for 15 days). The patient was discharged with aspirin therapy (50 mg/d). The family history was completely negative for venous and/or arterial thrombosis.
Laboratory investigations including prothrombin time, aPTT, fibrinogen, antithrombin, plasminogen, and antiphospholipid antibodies were performed as previously reported.7 Pooled normal plasma (reference plasma) was obtained from 44 healthy subjects of both sexes aged 20 to 65 years.
PC antigen and chromogenic activity were measured with the use of the enzyme-linked immunosorbent assay kit Asserachrom Protein C (kindly provided by Boehringer Mannheim, Milan, Italy) and Behrichrom Protein C (Behringwerke), respectively. Functional PC levels were also detected with the use of a clotting method by means of the Protein C Reagent kit (Behringwerke). To exclude dysfunctional PC molecules, additional immunologic and functional tests were performed as reported elsewhere.7
Total and free PS antigen and crossed immunoelectrophoretic assays were performed as previously reported.8 For the measurement of PS activity, the IL PS test (Instrumentation Laboratories) was used.
The presence of fast-acting inhibitors against APC was excluded by monitoring the hydrolysis of chromogenic substrate S-2366 after addition of APC to test plasma.
APC Resistance Test
The responsiveness of plasma to APC was measured as previously described5 and expressed as the ratio of two aPTTs, one in the presence of APC and one in its absence. The APC sensitivity ratio (APC-SR) was then normalized to the ratio obtained with a reference plasma (n-APC-SR). Resistance to APC is defined by n-APC-SR less than 0.84.3
The mean±SD APC-SR and the mean±SD n-APC-SR obtained from 40 normal subjects of both sexes were 2.64±0.3 and 1.02±0.13, respectively.
Genomic DNA was prepared from leukocytes by standard procedures. DNA analysis was performed as previously described3 with minor modifications. Briefly, a 220-bp fragment of exon 10/inton 10 of the factor V gene was amplified by polymerase chain reaction, with 5′-TGCCCAGTGCTTAACAAGACCA-3′ as a 5′ primer and 5′-CTTGAAGGAAA-TGCCCCATTA-3′ as a 3′ primer. Amplification involved 36 cycles of 91°C (40 seconds), 55°C (40 seconds), and 71°C (2 minutes) in the presence of 2 U Taq polymerase. Subsequently, the 220-bp fragment was digested during 16 hours by 0.4 U Mnl I at 37°C. Mnl I digests the 220-bp fragment of the normal factor V allele in three fragments of 37, 67, and 116 bp each. The factor V Leiden allele is cleaved in only two fragments of 67 and 153 bp. Finally, the digestion products were separated by electrophoresis on ethidium bromide–stained 2% agarose gels for 30 minutes at 150 V.
Tables 1 through 4⇓⇓⇓⇓ summarize the main laboratory findings and the results of the DNA analysis for the members of the three families investigated. The propositi exhibited the factor V Leiden mutation and a phenotype consistent with APC resistance. Several other family members were also found to be APC resistant. As can be seen in the family pedigrees in Fig 2⇓, the transmission of the defect is autosomal dominant. The penetrance of the defect seems to be quite high, at least for families B and C. In family B, 16 of 22 members investigated exhibited APC resistance. In addition, in this family some individuals (III-1, IV-1, IV-3) showed a very low n-APC-SR, and DNA analysis showed that these patients are homozygous for the Leiden mutation. In two of these cases the defect could be documented in both parents. It is interesting to note that in one instance (IV-1), one of the parents (III-1) was homozygous and the other (III-9) heterozygous for the mutation, but they were not consanguineous. In family C only the father of the propositus had a normal response to APC. Also in this case, the high prevalence can be explained by the concomitant presence of the defect in both patients I-1 and I-2 (Fig 2⇓ and Table 4⇓). Finally, in family A, 3 of 5 individuals studied exhibited APC resistance.
With regard to PC and PS levels, all APC-resistant individuals belonging to family A (Table 1⇑) had reduced PC clotting activity in the presence of normal antigen and chromogenic activity. The same was true for PS activity levels, which were reduced to half the normal value. Both total and free PS antigen were within the normal range. As can be seen in Tables 2⇑ and 3⇑, family B also presented discrepant clotting/immunologic levels for both PC and PS in all individuals except one (IV-2), who had normal PS activity. Finally, in family C, only some of the APC-resistant individuals presented discrepant values of PC and/or PS; the others showed normal levels.
Crossed immunoelectrophoresis of PC and PS in patients’ plasma failed to demonstrate any abnormal pattern. In addition, when PC or PS was isolated from plasma by immunoadsorption with monoclonal antibodies, normal clotting activity was detected (data not shown). No fast-acting inhibitor could be demonstrated in these patients. In addition, when PC and PS clotting tests were performed at higher dilutions of the plasma, higher values were obtained. All the other routine coagulation tests were found to be normal. No antiphospholipid antibody could be detected.
The recent discovery of a new mechanism that could explain inherited thrombophilia has been accepted with enthusiasm by physicians and researchers because thus far a large part of familial thrombosis could not be explained by deficiencies of the known anticoagulant proteins. The data from the literature seem to confirm that the prevalence of APC resistance among patients with a history of thrombosis is quite high, ranging between 21% and 64% on the basis of the selection criteria considered.4 5 6 In addition, the inheritance of the defect could be confirmed in the majority of cases, and the transmission was autosomal dominant. The mechanism of APC resistance has been clarified since Dahlback and Hildebrand2 suggested that APC resistance may be due to a selective defect in a cofactor function to APC of factor V. Bertina et al3 recently identified a mutation in blood coagulation factor V, ie, Arg506→Gln or factor V Leiden, that is responsible for resistance to APC. Two major studies5 6 have documented well that a poor anticoagulant response to APC is associated with venous thromboembolism. Acquired conditions interfering with the APC resistance test have not yet been clearly identified. We previously described a thrombophilic patient who presented a poor response to APC and the concomitant presence of antiphospolipid antibody.9 However, the family studies performed to this point have confirmed the inheritance of the defect in the majority of cases with APC resistance. Although venous thromboembolism seems to be the main clinical manifestation, at least one affected patient in each of the families reported here developed a juvenile stroke. Since stroke in juveniles and young adults has also been reported in association with other clotting inhibitor deficiencies,8 10 11 it seems of importance to report that this association is also found for inherited APC resistance. Recent reports seem to confirm this observation12 and a possible role of inherited APC resistance in arterial thrombosis.13 Halbmayer and coworkers12 reported a prevalence of APC resistance of 20% among patients with stroke. Genetic information was not given in this study, which had been performed before the report of factor V Leiden mutation as the cause of inherited APC resistance. Svensson and Dahlback6 described two patients with cerebral venous thrombosis and APC resistance. Therefore, it seems of importance to clarify adequately the nature of the occlusion, ie, arterial or venous, responsible for stroke. In a series of 16 consecutive young patients (aged <50 years) in Padua presenting with documented ischemic stroke due to arterial thrombosis, five (31.2%) showed APC resistance (Simioni et al, unpublished data, 1994). Genetic analysis in these patients and in their family members documented the factor V Leiden mutation. It is clear that case-control studies have to be performed to assess the true association between this abnormality and ischemic stroke.
In the last few years the role of triggering factors in the development of thrombotic manifestations in patients with inherited thrombophilia has been evaluated.14 15 In our patients a triggering event can be identified in approximately 50% of the symptomatic patients with inherited defects of clotting inhibitors. It is likely that triggering conditions might also be involved in the pathogenesis of thrombosis in patients with APC resistance. In the families described here, some thrombotic events had followed triggers such as surgery, trauma, or pregnancy. In contrast, childhood stroke in patient III-1 (family C) occurred spontaneously, and no other risk factors or triggering conditions could be detected. It is too early to speculate about the possible mechanisms whereby inherited APC resistance may cause arterial thrombosis and particularly ischemic stroke. Some information might come from the study of platelet factor V and/or from the clarification of the mechanism of factor Va/factor VIIIa inactivation in patients with the factor V Leiden mutation.
In agreement with earlier reports, the homozygous condition of APC resistance seems compatible with life and does not seem to be associated with severe or early thrombophilia, such as occurs in homozygous PC-deficient patients, who are generally symptomatic at young ages. Neonatal purpura fulminans also has not yet been reported in homozygous APC-resistant patients. In our cases, a 39-year-old homozygous woman, previously asymptomatic, had experienced only a superficial phlebitis of the leg in the postpartum period of the second pregnancy (after cesarean section). The other two homozygous patients are younger (a 17-year-old female and a 13-year-old male) and thus far asymptomatic. It should be established in the future whether homozygous patients will develop thrombotic episodes earlier in life than heterozygotes. The other six homozygous patients with APC resistance identified in Padua had a clinical presentation similar to that of heterozygotes. The reason for this is not known.
The laboratory detection of APC resistance is quite simple when the assay conditions have been well standardized. A complication may arise when a complete coagulation inhibitor study is performed in the plasma of APC-resistant patients. In fact, some spurious functional PS defects can be found that are dependent on the type of PS activity assay used, as demonstrated by Faioni et al.16 We confirmed these observations in the majority of the patients with APC resistance described in this report. However, it is not completely clear why some of the patients with APC resistance failed to present this pattern. These data clearly have to be taken into account when considering the diagnosis of hereditary PS defects. Another important point concerns the behavior of PC functional tests in the plasma of APC-resistant patients. Our results suggest that spurious slightly decreased PC clotting activity could be detected in the presence of normal antigen and chromogenic activity levels. The correlation between resistance to APC and reduced PC and PS anticoagulant activity in the affected members of families A and B is impressive. As is true for functional PS levels, not all the APC-resistant members of family C showed decreased levels of PC anticoagulant activity. The mechanism by which the factor V defect may influence both PS and PC clotting assays is probably the same because of the similarity of some of the steps in the assay procedures (eg, activation of PC by snake venom–, aPTT-, and/or prothrombin time–based methods). Particular attention must be paid to this problem when we attempt to identify truly dysfunctional PC molecules with these methods. In all our patients, the presence of antiphospholipid antibodies, which also may interfere in the protein C clotting assays,9 17 18 was excluded.
A final consideration deals with the therapeutic approach. There are no available data on this, but it is conceivable that heparin and oral anticoagulant treatment can be used in the treatment and secondary prevention of thrombosis in symptomatic APC-resistant patients. Because of the apparently high prevalence of the defect, we need to determine as soon as possible whether asymptomatic patients will need primary prevention. Although in this article we surmise a possible association between inherited APC resistance and stroke in juveniles and young adults, further investigation must be done to confirm this observation.
We thank Sabrina Gavasso (University of Padua) for her excellent technical assistance. Dr Simioni is particularly indebted to Emiel Wojcik (Thrombosis Research Center Leiden) for his most helpful comments. Furthermore, we want to thank Professor P.A. Battistella and Professor A.M. Laverda (Department of Pediatrics, University of Padua) for their help in the revision of the case report of family C, for their most valuable advice, and for having supplied and discussed the radiological images of their patient.
- Received October 6, 1994.
- Revision received January 26, 1995.
- Accepted January 26, 1995.
- Copyright © 1995 by American Heart Association
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