(Stroke. 1996;27:2005-2011.)
© 1996 American Heart Association, Inc.
Articles |
Impairments of the Protein C System and Fibrinolysis in Infection-Associated Stroke
the Departments of Neurology (R.F.M., S.F.A., M.F.) and Internal Medicine (R.B., F.P.Q., J.M.W.), University of Southern California School of Medicine, Los Angeles; and Scripps Research Institute, La Jolla, Calif (A.G., J.H.G., J.A.F.).
Correspondence to Richard F. Macko, MD, University of Maryland School of Medicine, Department of Neurology, 22 N Greene St, Baltimore, MD 21201-1595.
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Abstract |
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Background and Purpose Infection/inflammation appears to be an important predisposing risk factor for brain infarction, but little is known regarding underlying molecular mechanisms. We examined the hypothesis that patients with brain infarction preceded by infection/inflammation within 1 week could be identified by a distinctive procoagulant laboratory profile characterized by abnormalities in the protein C system and endogenous fibrinolysis.
Methods We performed a case-control study examining the relationship between preceding systemic infectious/inflammatory syndromes and selected immunohematologic variables in 36 patients with acute brain infarction and 81 control subjects (community control subjects [n=47] and hospitalized nonstroke neurological patient controls [n=34]).
Results The stroke group had a lower mean level of the circulating antithrombotic enzyme activated protein C (APC) (4.33±0.34% [log-transformed percentage of control value, mean±SD]) than community control subjects (4.51±0.27%, P<.02) or hospitalized neurological patient controls (4.57±0.31%, P<.005). The lowest circulating APC levels were found in the stroke group with antecedent infection/inflammation within 1 week preceding index brain infarction (4.23±0.4%, n=12). Within the stroke group, circulating APC levels were inversely related to IgG isotype anticardiolipin antibody titers (r=-.55, P<.001). Only the stroke group with infection/inflammation within 1 week had elevated plasma C4b binding protein compared with control subjects (141±61% versus 112±44%, P<.05). Stroke patients with antecedent infection/inflammation had a distinctively lower ratio of active tissue plasminogen activator to plasminogen activator inhibitor (0.11±0.04, n=9) than other stroke patients (0.19±0.06, n=9, P<.01) and control subjects (0.22±0.16, n=17, P<.02).
Conclusions Impairments in the protein C pathway and endogenous fibrinolysis may contribute to the increased risk for brain infarction after recent (
1 week) infection/inflammation. A decrease in the circulating anticoagulant APC may be related to elevated antiphospholipid antibody titers.
Key Words: blood coagulation • blood proteins • cerebral infarction • fibrinolysis • infection
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Systemic infection appears to be a common and important predisposing risk factor for brain infarction.1 2 3 4 Inflammation-mediated procoagulant changes have been hypothesized to link infection with stroke, but the hematologic mechanisms underlying this relationship have not been extensively investigated.2 3 5 In vitro, some inflammatory mediators have direct procoagulant effects on vascular endothelium, including downregulation of endothelial fibrinolytic capacity and impairment of the protein C antithrombotic mechanism.6 7 8 Similar disturbances in fibrinolysis and the protein C system are also described in sepsis-related disseminated intravascular coagulation9 10 11 and may function as hemostatic risk factors for brain infarction.12 13 14 15 The relationship between preceding infection/inflammation status and alterations in these key thrombosis regulatory systems has not been fully investigated in a stroke population.
The present case-control study examined the relationship between infection/inflammation and selected immunohematologic variables in subjects with acute brain infarction (within 4 days), community control subjects, and hospitalized patient controls who were admitted with a recent-onset noncerebrovascular neurological illness. In this population, we found a significant elevation in the prevalence of antecedent infection/inflammation in the stroke group exclusively within 1 week preceding the neurological event.16 Therefore, our aims were to identify coagulation regulatory system disturbances that might help to explain this apparent brief window of increased risk for stroke. This study examined the hypothesis that subjects with acute ischemic stroke preceded by systemic infection/inflammation within 1 week may present a distinct procoagulant laboratory profile characterized by impairments of the protein C antithrombotic pathway and endogenous fibrinolysis.
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Subjects and Methods |
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We evaluated 36 patients with acute ischemic stroke and 81 control subjects lacking history, signs, or symptoms of cerebrovascular disease. Patients admitted to LAC-USC with a diagnosis of acute ischemic stroke (<4 days since onset) and evaluated by the stroke unit (R.F.M., S.F.A., and/or M.F.) were consecutively entered. Stroke patients were evaluated a mean of 2.5±1 days (range, 1 to 4 days) after stroke onset. Community control subjects (n=47) consisted of ambulatory patients from the LAC-USC Hypertension Clinic or were from the LAC-USC Elderly Volunteer Group. Neurological patient controls (n=34) included patients hospitalized less than 4 days with a recent-onset noncerebrovascular neurological illness. Control patients were matched to stroke patients by age and seasonal time of enrollment. All subjects received a standardized evaluation including an infection/inflammation questionnaire.16 Stroke risk factors were characterized according to standard criteria.17 Ischemic strokes were classified as large-vessel occlusive disease, small-vessel or lacunar disease, cardioembolic, or other/undetermined.15 Exclusion criteria and clinical and demographic data for the stroke and control groups have been previously reported.16 Patients with excessive bruising or significant hematoma at a prior phlebotomy site were also excluded to avoid spurious abnormalities in plasma coagulation markers. The study was conducted according to guidelines of the LAC-USC Institutional Review Board, and all subjects gave informed consent.
Antecubital venipuncture with a 21-gauge butterfly needle was performed between 9 AM and 1 PM to minimize circadian variability in fibrinolytic measures.18 Venipuncture was performed without a tourniquet on reclining subjects to avoid augmenting TPA release, which is known to accompany venostasis, physical exertion, and orthostatic changes.19 20 The first 0.5 mL of blood was discarded, and plasma was used only if venous return was prompt throughout. All venipunctures were performed by the same two individuals (R.F.M. and S.F.A.). Blood samples were collected directly into precooled syringes, gently inverted five times, and placed immediately in an ice bath; platelet-poor plasma was prepared by centrifugation (4°C) within 30 minutes. Citrate-anticoagulated (0.11 mol/L, 9:1 vol/vol) venous blood for TPA activity determinations (Chromogenix) was immediately acidified within 60 seconds by addition of sodium acetate (0.5 mol/L, pH 4.2, 2:1 vol/vol) to prevent ongoing in vitro inactivation by PAI-121 and centrifuged for 10 minutes at 4000g.21 Although circulating PAI-1 levels are principally a function of endothelial release, platelet granules also contain PAI-1, and in vitro platelet activation may complicate plasma measures.22 We measured plasma PAI-1 activity (Chromogenix) from plasma prepared using a modification of the technique of Files et al23 to minimize in vitro platelet activation. Venous samples (4 mL) were collected into a precooled syringe containing 1 mL acid-citrate-dextrose, 80 µL aspirin (180 mg/mL ethanol), and 10 µL prostaglandin E1 (100 µL/mL ethanol).15 All samples were stored at -80°C until assayed in duplicate, with a maximum of one freeze-thaw cycle permitted.
Circulating APC, the principal antithrombotic enzyme in the protein C system, was directly measured by amidolytic assay after specific enzyme immunocapture.24 Specimens for APC determination were collected directly into a precooled syringe (1:9 vol/vol) containing citrate anticoagulant (0.11 mol/L) and benzamidine (0.5 mol/L), a reversible APC inhibitor.24 The mean plasma APC level as determined from individuals in the control population without infection/inflammation within 1 month was used as a reference value equal to 100%; all APC values were expressed as a percentage of this reference value. This control group was selected as a reference value to avoid any potential bias from the effects of preceding infection/inflammation on APC values. Total plasma protein S, protein C, and C4b binding protein antigen (C4bp, a main inhibitor of free protein S) were measured from citrate-anticoagulated specimens by enzyme immunoassays25 and expressed as a percentage of pooled plasma from healthy control subjects. FDD, an index of fibrin turnover, was measured by enzyme immunoassay (American Diagnostica) from citrate-anticoagulated samples.26 F1.2, an index of thrombin generation, was measured by enzyme immunoassay (Organon) from samples prepared in EDTA, heparin (20 U/mL) anticoagulants, and a proprietary sample treatment solution (Organon), which was added before freezing.27 We measured aCL types IgG and IgM using a solid-phase immunosorbent assay and reported as international phospholipid units (IU).29 Standardization included four known IgG and IgM serum samples, five normal serum samples, and five aCL-positive serum samples as secondary standards.
Unpaired two-tailed t tests were used for data analysis. Log-transformed data were used when necessary to best approximate the normal distribution. Regression analysis was used to examine correlations between hematologic variables.
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Results |
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Protein C System
The mean circulating APC level was significantly lower in the stroke group (4.33±0.34% [log-transformed percentage of control value], n=36) than in neurological patient controls (4.57±0.31%, n=34, P<.005) and community control subjects (4.51±0.27%, n=47, P<.02). In contrast, there were no significant differences in mean plasma protein C antigen levels between stroke patients and hospitalized neurological patient controls (97.3±31% versus 100±47% for pooled control subjects, P>.7) or community control subjects (91±32%, P>.3). We compared circulating APC levels in stroke patients with those in pooled control subjects without (n=60) and with infection/inflammation (n=21; mean, 10.6±10.8 days before enrollment) (Fig 1
). The lowest circulating APC levels were found in the stroke group with antecedent infection/inflammation within 1 week, which was significantly lower than in control subjects lacking infection/inflammation (P<.02) and somewhat (although not significantly) lower than in control subjects with infection/inflammation (P=.08). To place these data in perspective, circulating APC levels (when expressed as a percentage of control values) were 33% lower in the stroke group with recent infection/inflammation than in infection-free control subjects. Plasma protein C antigen levels were similar in the stroke groups with and without prior infection/inflammation within 1 week (92±36% versus 99±29%, P=.5).
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As a group, stroke patients had a mean plasma C4bp level similar to that in community control subjects (123.3±48 versus 111.4±46%, P>.2) and neurological patient controls (113±42%, P<.3). Only the stroke group with antecedent infection/inflammation within 1 week had greater C4bp antigen levels than pooled control subjects (Fig 2). Total protein S levels were similar in the stroke group (98±35%), community control subjects (99.4±35%, P>.8), and hospitalized neurological patient controls (102.6±35%, P>.5). There was a trend toward higher plasma total protein S antigen levels in the stroke group with antecedent infection/inflammation within 1 week than in other stroke patients (111.4±44 versus 91.2±29%, P=.11).
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Circulating APC levels were strongly positively correlated with protein C antigen levels in both the stroke (r=.68, P<.0001) (Fig 3) and pooled control groups (r=.66, P<.0001). In that subset of stroke patients in which fibrinolysis measures were performed (n=18), circulating APC levels were inversely related to PAI-1 activity levels (r=-.48, P<.05) (Fig 3). Simple regression analysis showed that circulating APC levels in pooled control subjects were weakly positively related (r=.22, P<.02) to the extent of thrombin generation as indicated by plasma F1.2 levels; however, multiple regression analysis revealed that only protein C antigen levels remained significantly related to circulating APC (n=81, r=.67, P<.0001). Circulating APC levels were also unrelated to plasma F1.2 in the stroke group (r=.098, P>.5). In the stroke group, simple regression analysis revealed that aCL IgG isotype titers were strongly inversely related to circulating APC levels (r=-.55, P<.001, n=34) (Fig 3) but had no significant relationship with protein C antigen levels (r=-.25, P>.16). As determined by multiple regression analysis, circulating APC levels in the stroke group remained significantly inversely related to aCL IgG titers (r=-.42, P<.002) and positively related to plasma protein C antigen levels (r=.53, P<.0001).
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Fibrinolysis and Fibrin Processing
Plasma TPA and PAI-1 activities were measured in a subset of stroke patients (n=18) and community control subjects (n=17). Stroke patients included subjects with (n=9) and without (n=9) antecedent infection/inflammation within 1 week. The stroke group with antecedent infection/inflammation within 1 week had significantly lower mean plasma TPA activity than community control subjects (2.1±0.6 versus 3.1±1 IU/mL, P<.02), and there was a trend toward higher levels of PAI-1 activity (21±7.6 versus 16.2±4.4 IU/mL, P=.051) (Fig 4). The stroke group without infection/inflammation within 1 week had mean plasma TPA (2.7±0.9 IU/mL, P>.3) and PAI-1 activity levels (15.4±7.5 IU/mL, P>.7) similar to those found in the control subjects. The stroke group with infection/inflammation within 1 week had a significantly lower ratio of active TPA to PAI-1 than the stroke group without infection/inflammation and the community control subjects (P<.01) (Fig 5).
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Stroke patients had significantly higher plasma FDD levels, regardless of infection/inflammation status, than control subjects (Fig 6). There was a trend toward higher mean FDD levels in the stroke group with recent infection/inflammation compared with other stroke patients (5.4±1.1 versus 4.8±0.7 log-transformed ng/mL, P=.1). To place these values in perspective, plasma FDD levels when expressed as nanograms per milliliter were 82% higher in the stroke group with recent infection/inflammation than in other stroke patients and 146% higher than in control subjects; mean plasma FDD levels in the stroke group lacking recent infection/inflammation were only 35% higher than those of control subjects. A separate analysis of all control subjects by sex revealed no significant differences between men and women in any of the hematologic variables studied (data not shown).
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Discussion |
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Altered hemostasis has been hypothesized to link infection with brain infarction, but only a few studies have examined circulating blood factors that may contribute to the underlying molecular mechanisms.2 3 4 5 Ameriso et al3 reported procoagulant abnormalities consisting of elevated plasma FDD and aCL titers in patients with brain infarction preceded by febrile infection within the preceding month. Our finding of elevated plasma FDD levels in stroke patients with antecedent infection/inflammation within 1 week is strikingly similar and suggests that these procoagulant alterations are often present in the 1 week preceding the neurological event. Interpretation of these procoagulant laboratory findings in infection-associated stroke is limited by the nonspecific nature of FDD and aCL elevations. The present study provides evidence that specific impairments in both the protein C antithrombotic system and the endogenous fibrinolysis system contribute to a procoagulant state underlying infection/inflammation-associated stroke.
Protein C circulates at 70 nmol/L in blood as an inactive zymogen.30 31 Normally, a small amount of the active antithrombotic protease APC circulates at approximately 40 pmol/L.24 APC functions by inhibiting activated coagulation factors Va and VIIIa and promoting fibrinolysis by inhibiting PAI-1.30 31 Prior studies in stroke populations have examined protein C antigen or protein C zymogen activity after in vitro addition of snake venom or other exogenous activators.14 32 33 34 In contrast, we directly measured the activity of the circulating antithrombotic enzyme24 and found low mean circulating APC but not protein C antigen after acute brain infarction. The stroke group had lower APC levels than neurological patient controls and community control subjects, demonstrating that circulating APC deficiency was a specific finding associated with brain infarction, not an incidental "acute-phase response" accompanying hospitalization with other noncerebrovascular neurological illness. Since we did not measure APC before the stroke, we cannot say for certain whether reductions in APC preceded or followed the neurological event.
Thrombomodulin is an integral endothelial membrane glycoprotein receptor that ablates the procoagulant functions of thrombin. Thrombin, bound to thrombomodulin, rapidly generates circulating APC from its zymogen form.30 31 35 In principle, low circulating APC may result from depletion of the protein C zymogen precursor, increased levels of circulating APC inhibitors, or reduced APC generating capacity due to either low levels of intravascular thrombin or reduced thrombomodulin at the endothelial surface. D'Angelo et al32 reported that low plasma protein C levels were related to poor outcome after stroke and hypothesized that protein C depletion occurred as a consequence of excessive thrombin generation and subsequent rapid APC clearance. These investigators found that stroke patients with lower protein C antigen levels had elevated levels of plasma fibrinopeptide A, indicating greater thrombin generation. In contrast, we found no relationship between levels of F1.2 and circulating APC in our stroke group, suggesting that low circulating APC levels occurred independent of the extent of thrombin generation, at least as judged by F1.2 levels.
We observed an intriguing relationship between positive infection/inflammation status and lower circulating APC levels in both stroke and control groups. Endotoxin and some inflammatory mediators, including interleukin-1 and tumor necrosis factor, are known to reduce thrombomodulin from the endothelial cell surface, resulting in downregulation of the thrombomodulin-dependent protein C activating capacity; this process likely contributes to inflammation-mediated disseminated intravascular coagulation.7 35 36 Our results are consistent with the hypothesis that inflammation associated with common infectious/inflammatory syndromes may contribute to lowered APC levels predisposing to stroke, in the absence of massive disseminated intravascular coagulation. In vitro, circulating APC promotes fibrinolysis by inhibiting PAI-1.30 Our findings in acute stroke patients that plasma PAI-1 activity levels were inversely related to circulating APC are consistent with these in vitro observations and suggest that coordinate impairment of fibrinolysis may accompany low circulating APC associated with brain infarction.
aCLs constitute a heterogeneous group of antibodies that bind phospholipid moieties and/or protein/phospholipid complexes and have been associated with thrombotic disorders, including stroke.37 38 Although specific hemostatic mechanisms relating aCL to stroke remain unclear, in vitro studies indicate that antiphospholipid antibodies may impair thrombomodulin-dependent protein C activation at the endothelial cell surface.39 40 Remarkably, we found a significant inverse relationship between levels of circulating APC and aCL titers in stroke patients, suggesting that aCL may have inhibitory effects on protein C activation in vivo. These correlative findings must be interpreted with caution because this study cannot distinguish whether aCL elevations were only a marker for recent infection/inflammation40 or directly contributed to reduction in APC generation. It is also possible that aCL or antiphospholipid antibodies impair the anticoagulant activity of APC, thereby contributing to increased risk of thrombosis.41 42
Other factors such as elevations in plasma C4bp may have further compromised the protein C system in infection-associated stroke patients. Deficiency of protein S, a cofactor optimizing the antithrombotic activity of free APC, is described as a predisposing risk factor for brain infarction.12 43 Protein S circulates in a free active form and in an inactive complex when bound to C4bp, a regulatory protein in the classic complement pathway.25 44 Elevations in C4bp, an acute-phase protein, often accompany inflammatory states and may foster an acquired free protein S deficiency.45 46 47 In the present study, only the stroke group with infection/inflammation within 1 week had C4bp levels significantly higher than control subjects. We did not directly measure free protein S. Thus, we interpret these elevations in C4bp as evidence of an inflammatory state but do not equate this to a relative free protein S deficiency condition, since it was recently shown that acute-phase increases in total C4bp do not usually involve increases of the functional ß-chain of C4bp or free protein S decreases.48
Numerous studies have demonstrated altered fibrin processing after stroke, but the causes of such abnormalities have not been well defined.15 49 50 Fibrinolysis is contingent not only on the availability of free TPA but also on the relative quantity of active PAI-1, its main circulating inhibitor.51 In endothelial cell cultures, endotoxin and some inflammatory mediators (eg, interleukin-1, tumor necrosis factor) downregulate fibrinolytic potential by reducing TPA production and increasing PAI-1 production.6 7 8 Similar disturbances of fibrinolysis regulation occur in disseminated intravascular coagulation and may be induced in healthy volunteers by parenteral endotoxin administration.9 52 53 We observed a striking association in our stroke group between positive antecedent infection/inflammation status, reduced TPA activity, and a lower ratio of active TPA to PAI-1, indicating reduced fibrinolytic capacity.51 These findings are consistent with the hypothesis that inflammation-mediated changes in vascular endothelium may contribute to impaired fibrinolytic capacity in the setting of brain infarction.
APC itself appears to have direct anti-inflammatory activity both in vitro and in vivo. When monocytes or macrophages were challenged with endotoxin, APC markedly attenuated the release of interleukin-8 and tumor necrosis factor.54 In a rat model of endotoxin-induced lung injury mediated by leukocytes, APC blunted leukocyte accumulation, inflammation, and tissue damage.55 In these and previous studies in baboons,35 36 the enzymatically active form of APC was required, and it was demonstrated that the anti-inflammatory activity of APC was independent of its anticoagulant activity. Currently it is speculated that the anti-inflammatory activity of APC is mediated by novel APC receptors on cell surfaces.54 56 Thus, the observed deficiency of circulating APC in stroke patients could compromise not only the antithrombotic mechanisms but also anti-inflammatory mechanisms.
This study is limited by small sample size and potential bias from sex ratio differences between groups. Although our stroke and control groups were matched by age and recruitment time, there was an unexpectedly lower ratio of men to women in the stroke group. We are uncertain as to the reason for this finding, since prior studies at LAC-USC had stroke populations with the expected slight male predominance.3 Although some hematologic variables, particularly fibrinolysis, are known to vary by sex, we believe that our results were unrelated to sex differences. Women are known to have lower PAI-1 activity and higher free-active TPA than men.57 Thus, the relative preponderance of women in our stroke group could have resulted in bias against our findings of impaired fibrinolysis in this population. However, a separate analysis in our pooled control subjects revealed no sex-related differences in any hematologic variables studied. Nonetheless, further studies are needed to explore sex-related differences in thrombosis regulatory mechanisms in stroke.
In summary, we found a distinctive procoagulant profile consisting of elevations in plasma FDD, C4bp levels, and a reduced fibrinolytic capacity in stroke patients with antecedent infection/inflammation within the previous week. Furthermore, we report that circulating APC deficiency may constitute a novel procoagulant state associated with stroke and provide evidence that the levels of this important antithrombotic enzyme may be related to precedent infection/inflammation status. Remarkably, the level of circulating APC was inversely correlated with aCL titer in stroke patients. The results of this study support the hypothesis that impairments in the protein C pathway and the endogenous fibrinolysis system may underlie important relationships between infection/inflammation and brain infarction. Systemic infections are common, and most are unrelated to clinical thrombotic events. Perhaps an exaggerated inflammatory-prothrombotic response as described in stroke-prone rats58 or hereditary factors59 may explain why inflammation may trigger thrombosis in some susceptible individuals. Further studies are needed to determine whether abnormalities of the protein C mechanisms and endogenous fibrinolysis represent enduring hematologic features after stroke or are related to vascular disease outcomes.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a National Stroke Association/CIBA-GEIGY Research Fellowship (R.F.M.) and by research grants NS-20989 (M.F.), P01NS31945 (M.F.), HL-15722 (R.F.M.), and CA-32197 (R.F.M.) from the National Institutes of Health, Bethesda, Md. We wish to thank Vicky L.Y. Wong for technical assistance.
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Footnotes |
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Reprint requests to Mark Fisher, MD, University of Southern California School of Medicine, Department of Neurology, 1333 San Pablo St, MCH 246, Los Angeles, CA 90033. E-mail mjfisher@hsc.usc.edu.
Received April 24, 1996; revision received June 20, 1996; accepted July 10, 1996.
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