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Altered influence of polymorphonuclear leukocytes on coagulation in acute ischemic stroke
Thrombosis Research, Vol. 76, No. 6, pp. 541-549, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved W9-3848/94 $6.00 + .oO
Pergamon
0049-3848(94)EOO169-3
ALTERED INFLUENCE OF POLYMORPHONUCLEAR LEUKOCYTES ON COAGULATION IN ACUTE ISCHEMIC STROKE. Armin J. &au, Tilo Graf, Werner Hacke Department of Neurology, University of Heidelberg, FRG
(Received 11 May 1994 by Editor E. Ernst; revised/accepted 10 October 1994)
Abstract Polymorphonuclear (PMN) and mononuclear (MN) leukocytes possess procoagulant and anticoagulant activity which could be involved in both formation and dissolution of thrombi. We investigated if coagulant properties of circulating leukocytes are altered in patients within three days after acute ischemic stroke (n=22) as compared to a control group (n=22) matched for sex and age. The recalcification time with autologous plasma did not differ between patients and control subjects. Circulating PMNs were procoagulant in all subjects, however, they were less procoagulant in patients (-18.1 [-13.4 - -22.81 % of control experiments; mean [95% confidence interval]) than in controls subjects (-31.9 [-27.4 - -36.41 %; p=O.O002). In contrast, MNs were similarly procoagulant in both groups. In the activated partial thromboplastin time (aPTI’), there was a non-significant trend to less procoagulant PMNs in patients (-6.7 [-5.1 - -8.21 %) than in control subjects (-8.4 [-6.4 - -10.51 %). The recalcification time with pooled human plasma showed similar results as with autologous plasma. The procoagulant activity of PMNs increased in follow-up measurements in patients. Upon stimulation with FMLP, the procoagulant activity of PMNs decreased in control subjects but did not change significantly in patients. In the acute stage after ischemic stroke, circulating PMNs exhibit a decreased capability to stimulate coagulation, a feature which reflects cell activation and which may be a reaction on thrombus formation and ischemic tissue damage.
Currently, an involvement of leukocytes in the pathogenesis of cerebrovascular ischemia is being discussed (1). Clinical studies have mainly focused on altered rheological properties and the release of cytotoxic substances as pathogenetic mechanisms involving leukocytes (2,3). However, polymorphonuclear (PMN) and mononuclear (MN) leukocytes can also influence the coagulation system (4) and this could potentially play a role in the generation and the consequences of cerebral thrombosis. Originally, the procoagulant activity of leukocytes was considered too weak to be significant, but later work showed that under appropriate conditions the contribution of leukocytes to thrombin generation is not neglible (4). In cooperation with a subset of T-lymphocytes (5), monocytes and Key words: Stroke, leukocytes, coagulation Corresponding author: Dr. Armin J Grau, Neurologische Klinik der Universitat Heidelberg, Im Neuenheimer Feld 400, D 69120 Heidelberg 541
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macrophages can develop considerable procoagulant activity by the release of tissue factor (TF) upon activation by endotoxin, immuncomplexes or other stimuli (6,7,8). In addition to TF, macrophages can produce all other clotting factors constituing the extrinsic pathway of coagulation (9). In a more rapid way, stimulated monocytes can directly activate factor X bound to the multifunctional adhesion receptor CD1 lb/CD18 on their cell surface and they express a factor V/Va-like binding site for factor Xa organizing a functional prothrombinase complex (10). Monocytes and macrophages also express sites for the assembly of factors ma/VIII and can thereby efficiently support factor X activation via the intrinsic pathway (11). The procoagulant effect of mononuclear cells was shown to be important in the pathogenesis of disseminated intravascular coagulation in septic shock (12). Dependent on their state of activation, macrophages also possess fibrinolytic activity by the release of a plasminogen activator (13). Mononuclear leukocytes - and to a lower degree PMNs - can support activated protein C-dependent clot lysis in the presence of plasminogen (14). Whole and intact PMNs from human subjects exhibit potent clot-promoting activity in vitro in cell concentrations normally found in vivo (15). On the other hand, human PMNs can develop fibrinolytic activity (16) by the release of a plasminogen activator (17) and of proteases, mainly elastase. PMN elastase modifies the fibrinolytic activity of plasminogen (18) and is able to cleave several clotting factors and fibrin, thus, exerting anticoagulant and fibrinolytic activity (19,20). “White thrombi” often contain leukocytes in concentrations which can not be explained simply as accidental enclosures (21) and leukocytes can be found in microthrombi in the brain after cerebral ischemia caused by large vessel occlusion (22 and Cervos-Navarro J, personal communication). Ongoing clot formation represents a chemotactic stimulus for PMNs (23) and activates the release of elastase (24). The presence of PMNs within thrombi could contribute to their disruption mechanically, by the release of fibrinolytic activity and by the phagocytosis and intracellular digestion of fibrin (25). Phagocytosis of fibrin by circulating PMNs could be demonstrated in patients with cerebral thrombosis (26). Under physiological conditions there appears to be a balance of procoagulant and anticoagulant activities of leukocytes. In acute cerebral ischemia, increased procoagulant properties of leukocytes could play a role in the initiation of thrombosis, whereas increased anticoagulant features could be a response to ongoing thrombus formation. This study was conducted in order to answer the question if and in which way coagulant properties of circulating leukocytes are altered in acute ischemic stroke. SUBJECTS AND METHODS We investigated 22 patients with ischemic stroke within three days after the onset of symptoms. The group consisted of 7 women and 15 men with an age of 63.6k12.2 years (mean&SD). In all patients a cerebral hemorhage was excluded by an early CT scan. Further exclusion criteria included trauma, surgery, or acute vascular diseases such as myocardial infarction within the last four weeks and treatment with drugs influencing the coagulation system (e.g. heparin or warfarin) or leukocyte function (e.g. steroids and non-steroidal antiinflammatory agents). None of the patients had a history of a recent infection. The control group consisted of 22 healthy subjects, 8 women and 14 men, with an age of 61.5f9.5 years. The protocol of this study was approved by the Institutional Review Committee of the University Hospital Heidelberg. Following consent, 20 ml of venous blood were drawn with minor stasis from an antecubital vein into plastic syringes with sodium citrate (1:9) as anticoagulant (Sarstedt, Ntimbrecht, Germany). In a first centrifugation step (170 g, 10 min), we gained platelet rich plasma (PRP) in order to minimize the content of contaminating platelets in leukocyte suspensions. Platelet poor plasma (PPP) was then won out of PRP (1500 g; 10 min). In the remainig blood, we substituted the volume of PRP by Dulbecco’s phosphate buffered saline (DPBS) without Ca++ and Mg++ (Sigma, Munich, Germany). In order to isolate leukocytes, dextran (2 ml of 6% dextran 500 in 0.85% NaCl; Pharmacia, Freiburg, Germany) was added to the the diluted blood. After
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sedimentation of the erythrocytes for 30 to 45 minutes, the leukocyte enriched supematant was layered on a Ficoll gradient (Biochrom, Berlin, Germany) and centrifuged for 20 minutes at 800 g. The PMN layer containing residual erythrocytes then underwent twice a hypotonic lysis with sterile destilled water for 20 seconds. The PMN and MN cell layers were washed in DPBS (170 g; 10 min) and finally the cells were resuspended in RPMI 1640 with 25 mM Hepes buffer and L-Glutamine (Gibco, Paisley, Scotland). Cell concentrations were adjusted to 25OO/ltl for PMNs and 1250&l for MNs (final concentrations in the clotting assays before addition of CaC12). None of the reagents contained more than 0.03 units/ml of endotoxin (USP-EE) as tested by a limulus assay (Pyroquant Diagnostik, Walldorf, Germany). The viability of cells was 2 98 % as evidenced by the trypan blue-exclusion method. The leukocyte suspensions contained 2.6~h1.8platelets/MN and 0.4ti.17 platelets/PMN (mean&SD), respectivly, without significant differences between groups. This contamination with platelets was accepted in order to avoid further washing procedures which may have led to cell activation. The contamination with PMNs in the MN cell suspension and with MNs in the PMN cell layer was 12 %, respectively, as evidenced microscopically. The MN cell suspensions consisted of 25+8 % (meatiSD; range lo42%) monocytes as demonstrated by alpha-naphtyl esterase staining. There were no differences between patients and controls. In order to test the influence of leukocytes on coagulation, routine assay systems were used. The recalcification time and the aPTI were done with autologous plasma diluted 1:2 either with leukocyte suspensions or with pure medium as control using a ball coagulometer (Amelung, Lemgo, Germany). Into plastic tubes, we first placed 50 ~1 plasma followed by 50 ~1 cell suspension or medium and finally 100 pl medium (recalcification time) or aPTT-reagent (ActinFS; Baxter, Unterschleissheim, Germany) were added. After allowing the constituents to warm up to 37°C the assays were started by the addition of 100 pl CaC12. All experiments were done in triplicate; the coefficient of variation was 0.06 on average in the recalcification time assays. The experiments were terminated within 3,5 hours after venipuncture. In smaller groups of subjects additional experiments were done in order to gain insight into mechanisms of altered leukocyte coagulant properties after stroke. Leukocytes were stimulated with N-formyl-methionin-leucine-phenylalanin (FMLP) ( 1O-7M final concentration) (Sigma, Munich, Germany) for 10 minutes at 37’C prior to the assays. In another subgroup of subjects, pooled human plasma (Baxter, Unterschleissheim, Germany) was used instead of autologous plasma. As most patients received treatment with heparin after their admission, follow-up experiments were only performed with heterologous plasma. In order to test the coagulant properties of the cell content, leukocytes were disrupted by 5 repeated freeze thaw cycles, and after a final ultracentrifugation (165000 g; 5 min), the supematant of lysed cells was investigated. Levels of unbound and active leukocyte elastase in the plasma free supematants of lysed cells were measured by a photometric assay (Kabi, Miinchen, Germany). Leukocyte, PMN and MN cell counts were done by Coulter counter (Hialeah, Florida, USA). We avoided calibrations of the results in clotting assays by standard curves due to the uncertainty about the relative contributions of different pathways in leukocyte related alterations of coagulation. Instead, results were expressed as differences in seconds and as percentages of control assays without leukocytes (10,15). Data are presented as mean and 95% confidence interval unless specifically mentioned. For statistical anlysis we used the Mann-Whitney U-test to evaluate the significance of differences between groups and the Wilcoxon signed rank test to analyze intraindividual differences. The statistical procedure was two-sided unless specifically mentioned. RESULTS
In preliminary experiments with plasma and leukocytes of healthy young volunteers the described assay yielded a cell concentration dependent shortening of the recalcification time by both PMNs and MNs (fig. 1). In experiments with patients and age and sex matched control
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subjects there was no difference in the recalcification time of autologous plasma diluted with medium (table 1). Upon addition of PMNs, the recalcification time was shortened in all control subjects and all but one of the patients. This shortening effect was significantly smaller in patients than in the control group as expressed in absolute numbers (seconds) (p=O.O14). As the most pronounced result, the decrease relative to individual control values showed a highly significant difference between groups with lower values in patients (p=O.O002) (table 1). This indicates a decreased procoagulant activity of PMNs after stroke as compared to the control group. The coagulant activity of MNs, however, did not differ between patients and control subjects (table 1). The aP’IT with autologous plasma diluted with medium tended to be shorter in o/o 1057 100 95
PMNs
,
90 85.
*
80. 75. 70. 65, 60. cells I ml
.5Fi-,313X106
,625x106
1,25x10'
2,5x106
5x106
%
MNs
-,156x10'
,313x106
,625x106
1,25x10'
2,5x106
FIG. 1. The influence of PMNs and with MNs on the recalcification time in 4 young and healthy volunteers. Results are expressed as percentages of control experiments without cells (mea&SD).
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patients (47.3 [44.5 - 50.21 s) than in control subjects (50.4 [46.8 - 53.91 s; p=O.OSS).PMNs from all subjects accelerated the aPTT but, as can be expected, their procoagulant effect was relativly small under conditions of already strongly stimulated coagulation. PMNs from patients tended to stimulate coagulation less than PMNs from control subjects, both in absolute numbers (-3.2 r-2.4 - -4.01 s in patients vs. -4.4 [-3.0 - -5.81 s in control subjects; p=O.O96) and related to individual control values (-6.7 r-3.0 - -5.81 % in patients vs. -8.4 [-6.4 - -10.51 % in the control group; p=O.15) but differences were not significant. This result shows the same tendency as findings in the recalcification time where differences were much more pronounced, however. MNs led to a minor increase of the aPlT, but there were no differences between groups (data not shown). In order to investigate if a plasmatic factor is responsible for the above differences between groups, we tested pooled plasma from healthy human subjects instead of autologous plasma. The procoagulant effect of PMNs in the recalcification time was stronger and the interindividual variability was smaller in pooled than in autologous plasma (table 2). The acceleration of the recalcification time by PMNs was again smaller in patients than in control subjects (p=O.O09). In patients a second test was done three to four days after admission. In all patients the procoagulant activity of PMNs increased during this period (p=O.OOSby Wilcoxon signed rank test). In pooled human plasma, the coagulant properties of MNs showed no difference between patients and control subjects, and those properties were not significantly altered in patients three to four days after admission. Upon stimulation with FMLP ( 10-7M), the procoagulant activity of PMNs in the recalcification time decreased in all but one of the patients (n=7) and in all but one of the control subjects (n=9); the interindividual variabilty of the responses to stimulation was considerable, however. In intraindividual comparisons, stimulation by FMLP led to a significant decrease of the procoagulant potential of PMNs only in the control group (-30.3 [-20.9 - -39.61 % (unstimulated) versus -15.1 r-1.0 - -29.11 % (FMLP), p=O.O15)but not in patients (-22.0 [-13.5 -30.51 % (unstimulated) versus -15.1 [-0.5 - -29.71 % (FML,P); p=O.18 ). In order to further elucidate potential mechanisms explaining the reduced coagulant potential of Table I Recalcification Time in Autologous Plasma with PMNs or MNs or with Medium as Control in Patients and Control Subjects. Parameter
control
Unit
Patients
Controls
n=22
n=22
S
365 [304 - 4251
S
-
p-value
377 [31 l- 4431
n.s.
-125 [-93 - -1571
p = 0.014
- 31.9 [-27.4 - -36.41
p = 0.0002
Differences from controls with PMNs
% with MNs
S
%
76 [-49 - -1021
-18.1 r-13.4 - -22.81 -
85 [-53 - -1171
-22.0 r-16.6 - -27.31
- 101 [-73 - -1291
n.s.
- 25.9 [-21.0 - -30.81
n.s.
Values represent mean and 95% confidence interval; P-values by Mann-Whitney U-test.
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Table II Recalcification Time in Heterologous Plasma with PMNs or MNs or with Medium as Control in Patients and Control Subjects and Follow-up Measurements in Patients. Parameter
control
Unit
S
Patients
Controls
n=lO
n=ll
p-value
421 [397 - 4461
420 [404 - 4361
n.s.
-43.7 [-36.9 - -50.51
p = 0.009
-37.4 [-34.7 - -40.21
n.s.
Differences from controls with PMNs
%
-33.4 [-29.8 - -36.91
after 3-4 days
%
-38.7 [-35.2 - -42.3]*
%
-37.5 [-30.2 - -44.71
%
-34.2 [-29.0 - -39.41
with MNs after 3-4 days
Values represent meanand 95% confidence interval; P-values by Mann-Whitney U-test. *Different from first measurements by Wilcoxon signed rank test (p=O.OOS). PMNs after stroke, we tested the hypothesis that the cytosol and the granules of PMNs after stroke contain more coagulation inhibiting activity and particularly more elastase. The supernatant of lysed PMNs did not prolong the recalcification time significantly more in patients (n=17, +93 [81 - 1041 %) than in control subjects (n=8, +85 [71 - 991 %). In the supematant of lysed PMNs, the activity of free elastase did not differ between patients (n=19, 60 [47 - 731 mu/107 PMNs) and the control group (n=8,67 [42 - 931 mu/107 PMNs); the supematant of MNs did not contain significant amounts of free elastase. Leukocyte counts were higher in patients (n=25, 9.6 [8.1 - 11.81 /nl) than in control subjects (n=18, 6.7 [5.9 - 7.61 /nl, p
DISCUSSION There is ample evidence that coagulation is activated in patients with acute ischemic stroke (27). Leukocytes can considerably modulate coagulation and leukocytes are activated after stroke as shown by rheological and other parameters (1,2,3). However, the coagulant potential of circulating leukocytes in cerebral ischemia had not been investigated, yet. As coagulant properties of leukocytes are altered upon stimulation, particular care was taken to minimize
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artificial cell activation and standardized working techniques were strictly maintained during all experiments. PMNs exhibited a decreased capability to stimulate coagulation in patients after stroke as measured by the unstimulated recalcification time. This finding could be caused by lower procoagulant activity but also by a higher release of anticoagulant activity by PMNs or both. In the aPTT the coagulant potential of PMNs was not strong enough to considerably modulate activated coagulation during the relativly short duration of the assays; however, results from a.PlT experiments again indicate a lower coagulation stimulating potential of PMNs after stroke. Procoagulant activity of PMNs seems to be mediated at least partly by binding sites for the prothrombinase complex (factor Va/factor Xa) on the cell membrane (28). These receptors for common pathway protease assembly could be down-regulated after stroke. PMN granules contain anticoagulant proteases, mainly elastase, and an augmented release of those enzymes in patients could also account for the detected differences. The results from clotting assays with lysed PMNs and from measurements of elastase content of PMNs do not provide evidence for a significantly increased anticoagulant potential inside cells in patients. However, PMNs may be primed in stroke patients and the release of anticoagulant activity may be faciliated during coagulation in our assays. Recently, we found increased levels of elastase alphat-proteinase inhibitor complexes in the plasma of patients after acute ischemic stroke, a result indicating an increased release of elastase by PMNs after cerebral ischemia in vivo (29). Upon stimulation with FMLP, the procoagulant potential of PMNs decreased in most subjects, however, this decrease was much stronger in controls than in patients. Therefore differences between groups in coagulant properties of PMNs disappeared upon stimulation with FMLP. This result indicates that PMNs after stroke are activated in terms of their coagulant properties and that they are more resistant to further stimulation. In patients, the procoagulant activity by PMNs increased 3 to 4 days after admission as compared to initial measurements. The coagulation stimulating capacity was still lower than in controls, however, the difference was not significant anymore. This result supports the hypothesis that altered coagulant properties represent at least partly a reaction of PMNs to cerebral thrombosis or to its consequences. Ongoing thrombus formation after stroke or pathways occuring in parallel with coagulation can activate PMNs (24), and altered coagulant properties of PMNs may partly be attributed to an acute phase reaction after stroke which gradually resolves during the first days after ictus. The question remains if the alteration of PMN coagulant properties is completly explained by an acute phase reaction or if a preexisting condition may also be relevant in this respect. An investigation of patients with stroke risk factors and of patients months after stroke may be helpful to solve this problem. It may be argued that lower procoagulant properties of single PMNs are counteracted by higher PMN counts after stroke. The physiological significance of coagulant properties of PMNs is not sufficiently known, especially not in the microcirculation where the coagulant features of single cells may be of particular significance. However, a reduced tendency to support fibrin generation may potentially have positive effects under low-flow conditions in ischemic tissue. Our study only investigated circulating PMNs whereas after acute ischemia a considerable number of activated leukocytes may be lost in the microcirculation. The relationship between leukocyte activation and fibrin generation in the microcirculation may be a rewarding subject of future investigations. In a study several years ago, phagocytosed fibrin was detected in circulating PMNs after stroke and the fibrinolytic activity of PMNs was discussed to be an important and protective mechanism in the sequelae of ischemic stroke (26). In our experiments we did not measure fibrinolytic activity of cells. However, if an increased release of proteases such as elastase causes the lower procoagulant activity of PMNs after stroke, this mechanism would also imply an increased thrombolytic potential of PMNs. On the other side, an augmented release of proteases by PMNs may also lead to injury of adjacent tissue, particularly of endothelial cells (30). Our experiments failed to show alterations of coagu!ant properties of circulating MNs after stroke, a finding which is not caused by differences in the composition of cell suspensions
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between groups. Increased coagulant activity of macrophages from carotid artery atheromas has recently been demonstrated (31). In addition, monocytes adherent to the endothelium may behave differently than circulating cells particularly since an engagement of the cell surface receptor CDllb/CD18 amplifies the induced TF response (32). Monocytes and macrophages could potentially contribute to the initiation of coagulation upon stimulation, for example during an infection, a potential risk factor for stroke (33,34). This study provides first evidence for an alteration of coagulant properties of PMNs after stroke. This deserves further investigation and particularly the fibrinolytic potential of leukocytes after stroke should be addressed in the future. REFERENCES 1. KOCHANEK, P.M., HALLENBECK, J.M. Polymorphonuclear leukocytes and monocytes/ macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 23, 1367-1379,1992. 2. MERCURI, M., CIUFFETTI, G., ROBINSON, M., TOOLE, J. Blood cell rheology in acute cerebral infarction. Stroke 20, 959-962, 1989. 3. GRAU, A.J., BERGER, E., SUNG K.L.P., SCHMID-SCHGNBEIN, G.W. Granulocyte adhesion, deformability, and superoxide formation in acute stroke. Stroke 23, 33-39, 1992. 4. LUSCHER, E.F. Activated leukocytes and the hemostatic system. Rev Infect Dis 9, S546S552, 1987. 5. EDWARDS, R.L., RICKLES, F.R. The role of human T cells (and T cell products) for monocyte tissue factor generation. J Immunol, 125, 606609, 1980. 6. EDWARDS, R.L., RICKLES, F.R., BOBROVE, A.M. Mononuclear cell tissue factor: Cell of origin and requirements for activation. Blood 54, 359-370, 1979. 7. SCHWARTZ, B.S., EDGINGTON, T.S. Immune complex-induced human monocyte procoagulant activity. I. A rapid unidirectional lymphocyte-instructed pathway. J Exp Med Z54, 892-906, 1981. 8. LEVY, G.A., SCHWARTZ, B.S., CURTISS, L.K., EDGINGTON, T.S. Plasma lipoprotein induction and suppression of the generation of cellular procoagulant activity in vitro. Requirements for cellular collaboration. J Clin Invest 67, 1614-1622, 1981. 9. OSTERUD, B., LINDAHL, U., SELJELID, R. Macrophages produce blood coagulation factors. FEBS Lett 120, 41-43, 1980. 10. ALTIERI, D.C. and EDGINGTON, T.S. Sequential receptor cascade for coagulation proteins on monocytes. Constitutive biosynthesis and functional prothrombinase activity of a membrane form of factor VfVa. J Biol Chem 264, 2969-2972, 1989. 11. MCGEE, M.P., LI, L.C. Functional difference between intrinsic and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages. J Biol Chem 266, 8079-8085,199l. 12. ANDERSEN, O.K., QISTERUD, B., GAUDERNACK, G., LUNDGREN, T.I., GIERCKSKY, K.E. Tissue thromboplastin generation in circulating mononuclear phagocytes and development of coagulation disorders during E. coli endotoxinaemia in pigs. Acta Chir Scand 151, 205-211,1985. 13. GORDON, S., UNKELESS, J.C., COHN, Z.A. Induction of macrophage plasminogen activator by endotoxin stimulation and phagocytosis. Evidence for a two-stage process. J Exp Med I40,995-1010,1974. 14. TAYLOR, F.B., LOCKHART, MS. Whole blood clot lysis: In vitro modulation by activated protein C. Thromb Res 37, 639-649, 1985. 15. SABA, H.I., HERION, J.C., WALKER, R.I., ROBERTS, H.R. The procoagulant activity of granulocytes. PSEBM Z42, 614-620, 1973. 16. RULOT, H. Intervention des leucocytes dans l’autolyse de la fibrine (fibrinolyse de dastre). Arch Int Physiol B&him,], 152-158, 1904. 17. GRANELLI-PIPERNO, A., VASSALLI, J.D., REICH, E. Secretion of plasminogen activator by human polymorphonuclear leukocytes. Modulation by glucocorticoids and other effecters. J Exp Med 146, 1693-1706,1977.
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18. MOROZ, L.A. Mini-plasminogen: A mechanism for leukocyte modulation of plasminogen activation by urokinase. Blood58, 97-104,198l. 19. PLOW, E.F., EDGINGTON, T.S. An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte proteases at physiologic PH. J Clin Invest 56, 30-38, 1975. 20. BROWER, M.S., WALZ, D.A., GARRY, K.E., FENTON II, J.W. Human neutrophil elastase alters human alpha-thrombin function: Limited proteolysis near the gamma-cleavage site results in decreased fibrinogen clotting and platelet-stimulatory activity. Blood 69, 813-819, 1987. 21. HENRY, R.L. Leukocytes and thrombosis. Thromb Diath Haemorrh 13, 35-46, 1965. 22. SAMPAOLO, S., CERVOS-NAVARRO, J., DJOUCHADAR, D., FIGOLS, J. Clinical and experimental evidence of microthrombosis in cerebral ischemia In: Cerebral ischemia and hemorheology . A Hartmann, W Kuschinsky (eds.), pp 386-393, Springer, Berlin-Heidelberg (1987). 23. MORIN, A., ARVIER, M.M., DOUTREMEPUICH, F., VIGNERON, C., Coagulation impact on chemotactic activity generation for polymorphonuclear leukocytes. Thromb Res 59, 979-984, 1990. 24. PLOW, E.F. Leukocyte elastase release during blood coagulation. A potential mechanism for activation of the alternative fibrinolytic pathway. J Clin Invest 69, 564-572, 1982. 25. RIDDLE, J.M., BARNHART, M.I. Ultrastructural study of fibrin dissolution via emigrated polymorphonuclear neutrophils. Am J Path 45, 805-823, 1964. 26. BARNHART, M.I. Importance of neutrophilic leukocytes in the resolution of fibrin. Fed Proc 24, 846-853,1965. 27. FISHER, M., FRANCIS R. Altered coagulation in cerebral ischemia. Platelet, thrombin, and plasmin activity. Arch Neurol47, 1075-1079, 1990. 28. TRACY, P.B., EIDE, L.L., MANN, K.G. Human prothrombinase complex assembly and function on isolated peripheral blood cell populations. J Biol Chem 260, 2119-2124, 1985. 29. GRAU, A., SEITZ, R., IMMEL, A., STEICHEN-WIEHN, C., HACKE, W. Increased levels of leukocyte elastase in ischemic stroke. Stroke 24, 166, 1993 (abstract). 30. SMEDLY, L.A., TONNESEN, M.G., SANDI-IAUS, R.A., HASLETT, C., GUTHRIE, L.A., JOHNSTON, R.B., HENSON, P.M., WORTHEN, G.S. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 77, 1233-1243, 1986. 31. TIPPING, P.G., MALLIAROS, J., HOLDSWORTH, S.R. Procoagulant activity expression by macrophages from atheromatous vascular plaques. Atherosclerosis 79,237-243, 1989. 32. FAN, S.T., EDGINGTON, T.S. Coupling of the adhesive receptor CDllb/CD18 to functional enhancement of effector macrophage tissue factor response. J Clin Invest 87, 50-57, 1991. 33. SYRJANEN, J., VALTONEN. V.V., IIVANAINEN, M., KASTE, M., HUTTUNEN, J.K. Preceding infection as an important risk factor for ischaemic brain infarction in young and middle aged patients. Br Med J 296,156-l 160,1988. 34. GRAU, A., BANERJEE, T., STEICHEN-WIEHN, C., MAIWALD, M., ROHLFS, M., SUHR, H., FIEHN, W., KONIG, J., HACKE, W. Preceding infection as a risk factor in cerebral ischemia. A case-control study. Stroke 24, 182, 1993 (abstract).
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ALTERED INFLUENCE OF POLYMORPHONUCLEAR LEUKOCYTES ON COAGULATION IN ACUTE ISCHEMIC STROKE. Armin J. &au, Tilo Graf, Werner Hacke Department of Neurology, University of Heidelberg, FRG
(Received 11 May 1994 by Editor E. Ernst; revised/accepted 10 October 1994)
Abstract Polymorphonuclear (PMN) and mononuclear (MN) leukocytes possess procoagulant and anticoagulant activity which could be involved in both formation and dissolution of thrombi. We investigated if coagulant properties of circulating leukocytes are altered in patients within three days after acute ischemic stroke (n=22) as compared to a control group (n=22) matched for sex and age. The recalcification time with autologous plasma did not differ between patients and control subjects. Circulating PMNs were procoagulant in all subjects, however, they were less procoagulant in patients (-18.1 [-13.4 - -22.81 % of control experiments; mean [95% confidence interval]) than in controls subjects (-31.9 [-27.4 - -36.41 %; p=O.O002). In contrast, MNs were similarly procoagulant in both groups. In the activated partial thromboplastin time (aPTI’), there was a non-significant trend to less procoagulant PMNs in patients (-6.7 [-5.1 - -8.21 %) than in control subjects (-8.4 [-6.4 - -10.51 %). The recalcification time with pooled human plasma showed similar results as with autologous plasma. The procoagulant activity of PMNs increased in follow-up measurements in patients. Upon stimulation with FMLP, the procoagulant activity of PMNs decreased in control subjects but did not change significantly in patients. In the acute stage after ischemic stroke, circulating PMNs exhibit a decreased capability to stimulate coagulation, a feature which reflects cell activation and which may be a reaction on thrombus formation and ischemic tissue damage.
Currently, an involvement of leukocytes in the pathogenesis of cerebrovascular ischemia is being discussed (1). Clinical studies have mainly focused on altered rheological properties and the release of cytotoxic substances as pathogenetic mechanisms involving leukocytes (2,3). However, polymorphonuclear (PMN) and mononuclear (MN) leukocytes can also influence the coagulation system (4) and this could potentially play a role in the generation and the consequences of cerebral thrombosis. Originally, the procoagulant activity of leukocytes was considered too weak to be significant, but later work showed that under appropriate conditions the contribution of leukocytes to thrombin generation is not neglible (4). In cooperation with a subset of T-lymphocytes (5), monocytes and Key words: Stroke, leukocytes, coagulation Corresponding author: Dr. Armin J Grau, Neurologische Klinik der Universitat Heidelberg, Im Neuenheimer Feld 400, D 69120 Heidelberg 541
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macrophages can develop considerable procoagulant activity by the release of tissue factor (TF) upon activation by endotoxin, immuncomplexes or other stimuli (6,7,8). In addition to TF, macrophages can produce all other clotting factors constituing the extrinsic pathway of coagulation (9). In a more rapid way, stimulated monocytes can directly activate factor X bound to the multifunctional adhesion receptor CD1 lb/CD18 on their cell surface and they express a factor V/Va-like binding site for factor Xa organizing a functional prothrombinase complex (10). Monocytes and macrophages also express sites for the assembly of factors ma/VIII and can thereby efficiently support factor X activation via the intrinsic pathway (11). The procoagulant effect of mononuclear cells was shown to be important in the pathogenesis of disseminated intravascular coagulation in septic shock (12). Dependent on their state of activation, macrophages also possess fibrinolytic activity by the release of a plasminogen activator (13). Mononuclear leukocytes - and to a lower degree PMNs - can support activated protein C-dependent clot lysis in the presence of plasminogen (14). Whole and intact PMNs from human subjects exhibit potent clot-promoting activity in vitro in cell concentrations normally found in vivo (15). On the other hand, human PMNs can develop fibrinolytic activity (16) by the release of a plasminogen activator (17) and of proteases, mainly elastase. PMN elastase modifies the fibrinolytic activity of plasminogen (18) and is able to cleave several clotting factors and fibrin, thus, exerting anticoagulant and fibrinolytic activity (19,20). “White thrombi” often contain leukocytes in concentrations which can not be explained simply as accidental enclosures (21) and leukocytes can be found in microthrombi in the brain after cerebral ischemia caused by large vessel occlusion (22 and Cervos-Navarro J, personal communication). Ongoing clot formation represents a chemotactic stimulus for PMNs (23) and activates the release of elastase (24). The presence of PMNs within thrombi could contribute to their disruption mechanically, by the release of fibrinolytic activity and by the phagocytosis and intracellular digestion of fibrin (25). Phagocytosis of fibrin by circulating PMNs could be demonstrated in patients with cerebral thrombosis (26). Under physiological conditions there appears to be a balance of procoagulant and anticoagulant activities of leukocytes. In acute cerebral ischemia, increased procoagulant properties of leukocytes could play a role in the initiation of thrombosis, whereas increased anticoagulant features could be a response to ongoing thrombus formation. This study was conducted in order to answer the question if and in which way coagulant properties of circulating leukocytes are altered in acute ischemic stroke. SUBJECTS AND METHODS We investigated 22 patients with ischemic stroke within three days after the onset of symptoms. The group consisted of 7 women and 15 men with an age of 63.6k12.2 years (mean&SD). In all patients a cerebral hemorhage was excluded by an early CT scan. Further exclusion criteria included trauma, surgery, or acute vascular diseases such as myocardial infarction within the last four weeks and treatment with drugs influencing the coagulation system (e.g. heparin or warfarin) or leukocyte function (e.g. steroids and non-steroidal antiinflammatory agents). None of the patients had a history of a recent infection. The control group consisted of 22 healthy subjects, 8 women and 14 men, with an age of 61.5f9.5 years. The protocol of this study was approved by the Institutional Review Committee of the University Hospital Heidelberg. Following consent, 20 ml of venous blood were drawn with minor stasis from an antecubital vein into plastic syringes with sodium citrate (1:9) as anticoagulant (Sarstedt, Ntimbrecht, Germany). In a first centrifugation step (170 g, 10 min), we gained platelet rich plasma (PRP) in order to minimize the content of contaminating platelets in leukocyte suspensions. Platelet poor plasma (PPP) was then won out of PRP (1500 g; 10 min). In the remainig blood, we substituted the volume of PRP by Dulbecco’s phosphate buffered saline (DPBS) without Ca++ and Mg++ (Sigma, Munich, Germany). In order to isolate leukocytes, dextran (2 ml of 6% dextran 500 in 0.85% NaCl; Pharmacia, Freiburg, Germany) was added to the the diluted blood. After
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sedimentation of the erythrocytes for 30 to 45 minutes, the leukocyte enriched supematant was layered on a Ficoll gradient (Biochrom, Berlin, Germany) and centrifuged for 20 minutes at 800 g. The PMN layer containing residual erythrocytes then underwent twice a hypotonic lysis with sterile destilled water for 20 seconds. The PMN and MN cell layers were washed in DPBS (170 g; 10 min) and finally the cells were resuspended in RPMI 1640 with 25 mM Hepes buffer and L-Glutamine (Gibco, Paisley, Scotland). Cell concentrations were adjusted to 25OO/ltl for PMNs and 1250&l for MNs (final concentrations in the clotting assays before addition of CaC12). None of the reagents contained more than 0.03 units/ml of endotoxin (USP-EE) as tested by a limulus assay (Pyroquant Diagnostik, Walldorf, Germany). The viability of cells was 2 98 % as evidenced by the trypan blue-exclusion method. The leukocyte suspensions contained 2.6~h1.8platelets/MN and 0.4ti.17 platelets/PMN (mean&SD), respectivly, without significant differences between groups. This contamination with platelets was accepted in order to avoid further washing procedures which may have led to cell activation. The contamination with PMNs in the MN cell suspension and with MNs in the PMN cell layer was 12 %, respectively, as evidenced microscopically. The MN cell suspensions consisted of 25+8 % (meatiSD; range lo42%) monocytes as demonstrated by alpha-naphtyl esterase staining. There were no differences between patients and controls. In order to test the influence of leukocytes on coagulation, routine assay systems were used. The recalcification time and the aPTI were done with autologous plasma diluted 1:2 either with leukocyte suspensions or with pure medium as control using a ball coagulometer (Amelung, Lemgo, Germany). Into plastic tubes, we first placed 50 ~1 plasma followed by 50 ~1 cell suspension or medium and finally 100 pl medium (recalcification time) or aPTT-reagent (ActinFS; Baxter, Unterschleissheim, Germany) were added. After allowing the constituents to warm up to 37°C the assays were started by the addition of 100 pl CaC12. All experiments were done in triplicate; the coefficient of variation was 0.06 on average in the recalcification time assays. The experiments were terminated within 3,5 hours after venipuncture. In smaller groups of subjects additional experiments were done in order to gain insight into mechanisms of altered leukocyte coagulant properties after stroke. Leukocytes were stimulated with N-formyl-methionin-leucine-phenylalanin (FMLP) ( 1O-7M final concentration) (Sigma, Munich, Germany) for 10 minutes at 37’C prior to the assays. In another subgroup of subjects, pooled human plasma (Baxter, Unterschleissheim, Germany) was used instead of autologous plasma. As most patients received treatment with heparin after their admission, follow-up experiments were only performed with heterologous plasma. In order to test the coagulant properties of the cell content, leukocytes were disrupted by 5 repeated freeze thaw cycles, and after a final ultracentrifugation (165000 g; 5 min), the supematant of lysed cells was investigated. Levels of unbound and active leukocyte elastase in the plasma free supematants of lysed cells were measured by a photometric assay (Kabi, Miinchen, Germany). Leukocyte, PMN and MN cell counts were done by Coulter counter (Hialeah, Florida, USA). We avoided calibrations of the results in clotting assays by standard curves due to the uncertainty about the relative contributions of different pathways in leukocyte related alterations of coagulation. Instead, results were expressed as differences in seconds and as percentages of control assays without leukocytes (10,15). Data are presented as mean and 95% confidence interval unless specifically mentioned. For statistical anlysis we used the Mann-Whitney U-test to evaluate the significance of differences between groups and the Wilcoxon signed rank test to analyze intraindividual differences. The statistical procedure was two-sided unless specifically mentioned. RESULTS
In preliminary experiments with plasma and leukocytes of healthy young volunteers the described assay yielded a cell concentration dependent shortening of the recalcification time by both PMNs and MNs (fig. 1). In experiments with patients and age and sex matched control
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subjects there was no difference in the recalcification time of autologous plasma diluted with medium (table 1). Upon addition of PMNs, the recalcification time was shortened in all control subjects and all but one of the patients. This shortening effect was significantly smaller in patients than in the control group as expressed in absolute numbers (seconds) (p=O.O14). As the most pronounced result, the decrease relative to individual control values showed a highly significant difference between groups with lower values in patients (p=O.O002) (table 1). This indicates a decreased procoagulant activity of PMNs after stroke as compared to the control group. The coagulant activity of MNs, however, did not differ between patients and control subjects (table 1). The aP’IT with autologous plasma diluted with medium tended to be shorter in o/o 1057 100 95
PMNs
,
90 85.
*
80. 75. 70. 65, 60. cells I ml
.5Fi-,313X106
,625x106
1,25x10'
2,5x106
5x106
%
MNs
-,156x10'
,313x106
,625x106
1,25x10'
2,5x106
FIG. 1. The influence of PMNs and with MNs on the recalcification time in 4 young and healthy volunteers. Results are expressed as percentages of control experiments without cells (mea&SD).
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patients (47.3 [44.5 - 50.21 s) than in control subjects (50.4 [46.8 - 53.91 s; p=O.OSS).PMNs from all subjects accelerated the aPTT but, as can be expected, their procoagulant effect was relativly small under conditions of already strongly stimulated coagulation. PMNs from patients tended to stimulate coagulation less than PMNs from control subjects, both in absolute numbers (-3.2 r-2.4 - -4.01 s in patients vs. -4.4 [-3.0 - -5.81 s in control subjects; p=O.O96) and related to individual control values (-6.7 r-3.0 - -5.81 % in patients vs. -8.4 [-6.4 - -10.51 % in the control group; p=O.15) but differences were not significant. This result shows the same tendency as findings in the recalcification time where differences were much more pronounced, however. MNs led to a minor increase of the aPlT, but there were no differences between groups (data not shown). In order to investigate if a plasmatic factor is responsible for the above differences between groups, we tested pooled plasma from healthy human subjects instead of autologous plasma. The procoagulant effect of PMNs in the recalcification time was stronger and the interindividual variability was smaller in pooled than in autologous plasma (table 2). The acceleration of the recalcification time by PMNs was again smaller in patients than in control subjects (p=O.O09). In patients a second test was done three to four days after admission. In all patients the procoagulant activity of PMNs increased during this period (p=O.OOSby Wilcoxon signed rank test). In pooled human plasma, the coagulant properties of MNs showed no difference between patients and control subjects, and those properties were not significantly altered in patients three to four days after admission. Upon stimulation with FMLP ( 10-7M), the procoagulant activity of PMNs in the recalcification time decreased in all but one of the patients (n=7) and in all but one of the control subjects (n=9); the interindividual variabilty of the responses to stimulation was considerable, however. In intraindividual comparisons, stimulation by FMLP led to a significant decrease of the procoagulant potential of PMNs only in the control group (-30.3 [-20.9 - -39.61 % (unstimulated) versus -15.1 r-1.0 - -29.11 % (FMLP), p=O.O15)but not in patients (-22.0 [-13.5 -30.51 % (unstimulated) versus -15.1 [-0.5 - -29.71 % (FML,P); p=O.18 ). In order to further elucidate potential mechanisms explaining the reduced coagulant potential of Table I Recalcification Time in Autologous Plasma with PMNs or MNs or with Medium as Control in Patients and Control Subjects. Parameter
control
Unit
Patients
Controls
n=22
n=22
S
365 [304 - 4251
S
-
p-value
377 [31 l- 4431
n.s.
-125 [-93 - -1571
p = 0.014
- 31.9 [-27.4 - -36.41
p = 0.0002
Differences from controls with PMNs
% with MNs
S
%
76 [-49 - -1021
-18.1 r-13.4 - -22.81 -
85 [-53 - -1171
-22.0 r-16.6 - -27.31
- 101 [-73 - -1291
n.s.
- 25.9 [-21.0 - -30.81
n.s.
Values represent mean and 95% confidence interval; P-values by Mann-Whitney U-test.
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Table II Recalcification Time in Heterologous Plasma with PMNs or MNs or with Medium as Control in Patients and Control Subjects and Follow-up Measurements in Patients. Parameter
control
Unit
S
Patients
Controls
n=lO
n=ll
p-value
421 [397 - 4461
420 [404 - 4361
n.s.
-43.7 [-36.9 - -50.51
p = 0.009
-37.4 [-34.7 - -40.21
n.s.
Differences from controls with PMNs
%
-33.4 [-29.8 - -36.91
after 3-4 days
%
-38.7 [-35.2 - -42.3]*
%
-37.5 [-30.2 - -44.71
%
-34.2 [-29.0 - -39.41
with MNs after 3-4 days
Values represent meanand 95% confidence interval; P-values by Mann-Whitney U-test. *Different from first measurements by Wilcoxon signed rank test (p=O.OOS). PMNs after stroke, we tested the hypothesis that the cytosol and the granules of PMNs after stroke contain more coagulation inhibiting activity and particularly more elastase. The supernatant of lysed PMNs did not prolong the recalcification time significantly more in patients (n=17, +93 [81 - 1041 %) than in control subjects (n=8, +85 [71 - 991 %). In the supematant of lysed PMNs, the activity of free elastase did not differ between patients (n=19, 60 [47 - 731 mu/107 PMNs) and the control group (n=8,67 [42 - 931 mu/107 PMNs); the supematant of MNs did not contain significant amounts of free elastase. Leukocyte counts were higher in patients (n=25, 9.6 [8.1 - 11.81 /nl) than in control subjects (n=18, 6.7 [5.9 - 7.61 /nl, p
DISCUSSION There is ample evidence that coagulation is activated in patients with acute ischemic stroke (27). Leukocytes can considerably modulate coagulation and leukocytes are activated after stroke as shown by rheological and other parameters (1,2,3). However, the coagulant potential of circulating leukocytes in cerebral ischemia had not been investigated, yet. As coagulant properties of leukocytes are altered upon stimulation, particular care was taken to minimize
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artificial cell activation and standardized working techniques were strictly maintained during all experiments. PMNs exhibited a decreased capability to stimulate coagulation in patients after stroke as measured by the unstimulated recalcification time. This finding could be caused by lower procoagulant activity but also by a higher release of anticoagulant activity by PMNs or both. In the aPTT the coagulant potential of PMNs was not strong enough to considerably modulate activated coagulation during the relativly short duration of the assays; however, results from a.PlT experiments again indicate a lower coagulation stimulating potential of PMNs after stroke. Procoagulant activity of PMNs seems to be mediated at least partly by binding sites for the prothrombinase complex (factor Va/factor Xa) on the cell membrane (28). These receptors for common pathway protease assembly could be down-regulated after stroke. PMN granules contain anticoagulant proteases, mainly elastase, and an augmented release of those enzymes in patients could also account for the detected differences. The results from clotting assays with lysed PMNs and from measurements of elastase content of PMNs do not provide evidence for a significantly increased anticoagulant potential inside cells in patients. However, PMNs may be primed in stroke patients and the release of anticoagulant activity may be faciliated during coagulation in our assays. Recently, we found increased levels of elastase alphat-proteinase inhibitor complexes in the plasma of patients after acute ischemic stroke, a result indicating an increased release of elastase by PMNs after cerebral ischemia in vivo (29). Upon stimulation with FMLP, the procoagulant potential of PMNs decreased in most subjects, however, this decrease was much stronger in controls than in patients. Therefore differences between groups in coagulant properties of PMNs disappeared upon stimulation with FMLP. This result indicates that PMNs after stroke are activated in terms of their coagulant properties and that they are more resistant to further stimulation. In patients, the procoagulant activity by PMNs increased 3 to 4 days after admission as compared to initial measurements. The coagulation stimulating capacity was still lower than in controls, however, the difference was not significant anymore. This result supports the hypothesis that altered coagulant properties represent at least partly a reaction of PMNs to cerebral thrombosis or to its consequences. Ongoing thrombus formation after stroke or pathways occuring in parallel with coagulation can activate PMNs (24), and altered coagulant properties of PMNs may partly be attributed to an acute phase reaction after stroke which gradually resolves during the first days after ictus. The question remains if the alteration of PMN coagulant properties is completly explained by an acute phase reaction or if a preexisting condition may also be relevant in this respect. An investigation of patients with stroke risk factors and of patients months after stroke may be helpful to solve this problem. It may be argued that lower procoagulant properties of single PMNs are counteracted by higher PMN counts after stroke. The physiological significance of coagulant properties of PMNs is not sufficiently known, especially not in the microcirculation where the coagulant features of single cells may be of particular significance. However, a reduced tendency to support fibrin generation may potentially have positive effects under low-flow conditions in ischemic tissue. Our study only investigated circulating PMNs whereas after acute ischemia a considerable number of activated leukocytes may be lost in the microcirculation. The relationship between leukocyte activation and fibrin generation in the microcirculation may be a rewarding subject of future investigations. In a study several years ago, phagocytosed fibrin was detected in circulating PMNs after stroke and the fibrinolytic activity of PMNs was discussed to be an important and protective mechanism in the sequelae of ischemic stroke (26). In our experiments we did not measure fibrinolytic activity of cells. However, if an increased release of proteases such as elastase causes the lower procoagulant activity of PMNs after stroke, this mechanism would also imply an increased thrombolytic potential of PMNs. On the other side, an augmented release of proteases by PMNs may also lead to injury of adjacent tissue, particularly of endothelial cells (30). Our experiments failed to show alterations of coagu!ant properties of circulating MNs after stroke, a finding which is not caused by differences in the composition of cell suspensions
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between groups. Increased coagulant activity of macrophages from carotid artery atheromas has recently been demonstrated (31). In addition, monocytes adherent to the endothelium may behave differently than circulating cells particularly since an engagement of the cell surface receptor CDllb/CD18 amplifies the induced TF response (32). Monocytes and macrophages could potentially contribute to the initiation of coagulation upon stimulation, for example during an infection, a potential risk factor for stroke (33,34). This study provides first evidence for an alteration of coagulant properties of PMNs after stroke. This deserves further investigation and particularly the fibrinolytic potential of leukocytes after stroke should be addressed in the future. REFERENCES 1. KOCHANEK, P.M., HALLENBECK, J.M. Polymorphonuclear leukocytes and monocytes/ macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 23, 1367-1379,1992. 2. MERCURI, M., CIUFFETTI, G., ROBINSON, M., TOOLE, J. Blood cell rheology in acute cerebral infarction. Stroke 20, 959-962, 1989. 3. GRAU, A.J., BERGER, E., SUNG K.L.P., SCHMID-SCHGNBEIN, G.W. Granulocyte adhesion, deformability, and superoxide formation in acute stroke. Stroke 23, 33-39, 1992. 4. LUSCHER, E.F. Activated leukocytes and the hemostatic system. Rev Infect Dis 9, S546S552, 1987. 5. EDWARDS, R.L., RICKLES, F.R. The role of human T cells (and T cell products) for monocyte tissue factor generation. J Immunol, 125, 606609, 1980. 6. EDWARDS, R.L., RICKLES, F.R., BOBROVE, A.M. Mononuclear cell tissue factor: Cell of origin and requirements for activation. Blood 54, 359-370, 1979. 7. SCHWARTZ, B.S., EDGINGTON, T.S. Immune complex-induced human monocyte procoagulant activity. I. A rapid unidirectional lymphocyte-instructed pathway. J Exp Med Z54, 892-906, 1981. 8. LEVY, G.A., SCHWARTZ, B.S., CURTISS, L.K., EDGINGTON, T.S. Plasma lipoprotein induction and suppression of the generation of cellular procoagulant activity in vitro. Requirements for cellular collaboration. J Clin Invest 67, 1614-1622, 1981. 9. OSTERUD, B., LINDAHL, U., SELJELID, R. Macrophages produce blood coagulation factors. FEBS Lett 120, 41-43, 1980. 10. ALTIERI, D.C. and EDGINGTON, T.S. Sequential receptor cascade for coagulation proteins on monocytes. Constitutive biosynthesis and functional prothrombinase activity of a membrane form of factor VfVa. J Biol Chem 264, 2969-2972, 1989. 11. MCGEE, M.P., LI, L.C. Functional difference between intrinsic and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages. J Biol Chem 266, 8079-8085,199l. 12. ANDERSEN, O.K., QISTERUD, B., GAUDERNACK, G., LUNDGREN, T.I., GIERCKSKY, K.E. Tissue thromboplastin generation in circulating mononuclear phagocytes and development of coagulation disorders during E. coli endotoxinaemia in pigs. Acta Chir Scand 151, 205-211,1985. 13. GORDON, S., UNKELESS, J.C., COHN, Z.A. Induction of macrophage plasminogen activator by endotoxin stimulation and phagocytosis. Evidence for a two-stage process. J Exp Med I40,995-1010,1974. 14. TAYLOR, F.B., LOCKHART, MS. Whole blood clot lysis: In vitro modulation by activated protein C. Thromb Res 37, 639-649, 1985. 15. SABA, H.I., HERION, J.C., WALKER, R.I., ROBERTS, H.R. The procoagulant activity of granulocytes. PSEBM Z42, 614-620, 1973. 16. RULOT, H. Intervention des leucocytes dans l’autolyse de la fibrine (fibrinolyse de dastre). Arch Int Physiol B&him,], 152-158, 1904. 17. GRANELLI-PIPERNO, A., VASSALLI, J.D., REICH, E. Secretion of plasminogen activator by human polymorphonuclear leukocytes. Modulation by glucocorticoids and other effecters. J Exp Med 146, 1693-1706,1977.
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18. MOROZ, L.A. Mini-plasminogen: A mechanism for leukocyte modulation of plasminogen activation by urokinase. Blood58, 97-104,198l. 19. PLOW, E.F., EDGINGTON, T.S. An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte proteases at physiologic PH. J Clin Invest 56, 30-38, 1975. 20. BROWER, M.S., WALZ, D.A., GARRY, K.E., FENTON II, J.W. Human neutrophil elastase alters human alpha-thrombin function: Limited proteolysis near the gamma-cleavage site results in decreased fibrinogen clotting and platelet-stimulatory activity. Blood 69, 813-819, 1987. 21. HENRY, R.L. Leukocytes and thrombosis. Thromb Diath Haemorrh 13, 35-46, 1965. 22. SAMPAOLO, S., CERVOS-NAVARRO, J., DJOUCHADAR, D., FIGOLS, J. Clinical and experimental evidence of microthrombosis in cerebral ischemia In: Cerebral ischemia and hemorheology . A Hartmann, W Kuschinsky (eds.), pp 386-393, Springer, Berlin-Heidelberg (1987). 23. MORIN, A., ARVIER, M.M., DOUTREMEPUICH, F., VIGNERON, C., Coagulation impact on chemotactic activity generation for polymorphonuclear leukocytes. Thromb Res 59, 979-984, 1990. 24. PLOW, E.F. Leukocyte elastase release during blood coagulation. A potential mechanism for activation of the alternative fibrinolytic pathway. J Clin Invest 69, 564-572, 1982. 25. RIDDLE, J.M., BARNHART, M.I. Ultrastructural study of fibrin dissolution via emigrated polymorphonuclear neutrophils. Am J Path 45, 805-823, 1964. 26. BARNHART, M.I. Importance of neutrophilic leukocytes in the resolution of fibrin. Fed Proc 24, 846-853,1965. 27. FISHER, M., FRANCIS R. Altered coagulation in cerebral ischemia. Platelet, thrombin, and plasmin activity. Arch Neurol47, 1075-1079, 1990. 28. TRACY, P.B., EIDE, L.L., MANN, K.G. Human prothrombinase complex assembly and function on isolated peripheral blood cell populations. J Biol Chem 260, 2119-2124, 1985. 29. GRAU, A., SEITZ, R., IMMEL, A., STEICHEN-WIEHN, C., HACKE, W. Increased levels of leukocyte elastase in ischemic stroke. Stroke 24, 166, 1993 (abstract). 30. SMEDLY, L.A., TONNESEN, M.G., SANDI-IAUS, R.A., HASLETT, C., GUTHRIE, L.A., JOHNSTON, R.B., HENSON, P.M., WORTHEN, G.S. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 77, 1233-1243, 1986. 31. TIPPING, P.G., MALLIAROS, J., HOLDSWORTH, S.R. Procoagulant activity expression by macrophages from atheromatous vascular plaques. Atherosclerosis 79,237-243, 1989. 32. FAN, S.T., EDGINGTON, T.S. Coupling of the adhesive receptor CDllb/CD18 to functional enhancement of effector macrophage tissue factor response. J Clin Invest 87, 50-57, 1991. 33. SYRJANEN, J., VALTONEN. V.V., IIVANAINEN, M., KASTE, M., HUTTUNEN, J.K. Preceding infection as an important risk factor for ischaemic brain infarction in young and middle aged patients. Br Med J 296,156-l 160,1988. 34. GRAU, A., BANERJEE, T., STEICHEN-WIEHN, C., MAIWALD, M., ROHLFS, M., SUHR, H., FIEHN, W., KONIG, J., HACKE, W. Preceding infection as a risk factor in cerebral ischemia. A case-control study. Stroke 24, 182, 1993 (abstract).