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Prepublished online as a Blood First Edition Paper on April 10, 2003; DOI 10.1182/blood-2003-01-0324. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-01-0324v1 102/3/953    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gruber, A. Articles by Hanson, S. R. Search for Related Content PubMed PubMed Citation Articles by Gruber, A. Articles by Hanson, S. R. Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 August 2003, Vol. 102, No. 3, pp. 953-955 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Brief report Factor XI–dependence of surface- and tissue factor–initiated thrombus propagation in primates András Gruber, and Stephen R. Hanson From the Department of Biomedical Engineering at Georgia Institute of Technology & Emory University School of Medicine, Atlanta, GA; and the Yerkes National Primate Research Center, Atlanta, GA    Abstract Top Abstract Introduction Study design Results and discussion References   Thrombin, generated through activation of factor XI (FXI) and/or tissue factor (TF)–factor VIIa, is essential for thrombosis and hemostasis. We investigated the role of FXI-dependent thrombus propagation under arterial flow conditions producing rapid thrombus growth that, after the initiation phase, could limit the availability of TF at the blood/thrombus interface. Thrombosis was initiated by knitted dacron or TF-presenting teflon grafts deployed into arteriovenous shunts in baboons treated with antihuman FXI antibody (aFXI). Although aFXI did not prevent thrombus initiation, it markedly reduced intraluminal thrombus growth on both surfaces. The antithrombotic effect of aFXI was comparable with that of heparin at doses that significantly prolonged the partial thromboplastin time (APTT), prothrombin time (PT), and bleeding time (BT). aFXI also prolonged the APTT, but the PT and BT were unaffected. Thus, antithrombotic targeting of FXI might inhibit thrombosis with relatively modest hemostatic impairment versus strategies targeting other coagulation factors.    Introduction Top Abstract Introduction Study design Results and discussion References   Thrombin generation is initiated by exposure of blood to activated coagulation enzymes, tissue factor (TF), and/or foreign surfaces, and can be amplified through feedback activation of factor XI (FXI) and factor VII in the presence of platelets and TF, respectively.1 Thrombosis after knee replacement depends, in part, on extrinsic coagulation mechanisms that bypass FXI.2 However, the pathogenesis of thrombosis might be different in other cases. For example, large thrombi, which extend into the blood flow field, grow at the blood/thrombus interface that is removed from the initiating procoagulant factors present in the vessel wall. Hypothetically, the rapid radial and distal extension of such thrombi could depend importantly upon the activation of FXI, delivered continuously to the thrombus under blood flow. We therefore studied the effects of inhibition of FXI on the initiation and propagation of thrombi in primate arterial flow models, which produce robust thrombus growth.    Study design Top Abstract Introduction Study design Results and discussion References   In 35 nonterminal experiments, 8 nonanesthetized baboons (9-12 kg) were used. For in vivo anticoagulation, intravenous porcine heparin (1000 U/mL; Wyeth-Ayerst, Pearl River, NY) or affinity-depleted monospecific antihuman FXI polyclonal goat antibody (aFXI; GAFXI-AP, 5 mg/mL; Affinity Biologicals, Hamilton, ON, Canada) were used at 50 U/kg bolus plus infusion of 50 U/kg/h for 70 minutes and at 16 to 50 mg/kg bolus, respectively. Partial thromboplastin time (APTT) and prothrombin time (PT) were monitored using FDA-approved point-of-care diagnostic tests (Rapidpoint Coag, APTT and PT-One test cards; Bayer Diagnostics, Tarrytown, NY) as recommended, except 3.8% citrate was used as an anticoagulant. Template bleeding time (BT) (Surgicutt; ITC, Edison, NJ) was measured before and repeatedly during anticoagulation. The antithrombotic effects of heparin or aFXI were studied using 2 different thrombogenic devices deployed for 60 minutes into surgically placed high flow chronic arteriovenous (AV) shunts.3,4 In one setting, the device was composed of a 20-mm long dacron graft segment (0.25 mL) and a silicone extension chamber (1.3 mL) (DG), essentially as described.3 In another setting, the dacron/chamber was replaced with 20-mm long (0.25 mL) ringed expanded polytetrafluoroethylene (ePTFE, teflon) graft segments (WL Gore, Newark, DE) in the shunt. The hypothrombogenic ePTFE graft was converted into an acutely thrombogenic TF-dependent device (TG) for testing heparin and aFXI as follows. The ePTFE was wetted with ethanol, which was displaced by saline, followed by filling of the pores with thromboplastin (1:10 Innovin in saline; Dade Behring, Deerfield, IL) via transgraft perfusion until the reagent entered the graft lumen through the wall. The graft material retained approximately 0.2 µL/mm2 thromboplastin. Prior to deployment, the TG was rinsed by passing 50 mL saline through the lumen. In order to limit the time of blood exposure to TF retained in the porous graft wall, the reagent content of the pores was flushed into the blood stream starting at 10 minutes after the start of flow by transgraft perfusion of saline at a rate of 2.4 µL/mm2 graft surface/min using a pump. Blood flow was kept at 100 mL/min by proximal clamping. Thrombogenesis was assessed by measuring radiolabeled fibrin and platelet contents of the thrombogenic devices, as described.3,4 In brief, the terminal fibrin content of graft/chamber thrombi (fibrin deposition) was determined by direct 125Iodine-labeled fibrin counting. The number of deposited platelets in 35-cm long 4-mm internal diameter shunt segments that incorporated the devices and associated thrombi was quantified by 111Indium-labeled platelet imaging with 5-minute data acquisition periods. Net platelet accumulation rate (NPAR) was calculated as the change in the platelet content of the device within one period. Experiments included at least 1 control and 1 heparin study, and 3 aFXI studies performed in each of the 6 animals that were used for comparative analyses. The thrombogenicity of native ePTFE grafts was tested in 5 animals. Results are given as the mean ± SD, and as the percentage of untreated controls where appropriate. Results were compared using the t test; P < .05 was considered statistically significant.    Results and discussion Top Abstract Introduction Study design Results and discussion References   Since the point-of-care PT and APTT cards use dry chemistry, these tests were not affected by sample dilution. The use of 3.8% citrate in this test resulted in somewhat longer than typical PT values. High-dose heparin produced hemostatic impairment consistent with expectations based on human experience.5,6 The APTT (seconds) was prolonged from 28 ± 8 to 105 ± 54 (P < .001), the BT (minute) from 4.0 ± 1.0 to 5.6 ± 1.2 (P < .01), and the PT (seconds) from 21 ± 5 to 51 ± 17 (P < .001). Coagulation tests following aFXI administration suggested impairment of the intrinsic pathway, as expected. aFXI pretreatment prolonged the APTT to 89 ± 58 (P < .001) for at least 25 hours but BT (4.1 ± 1.2) and PT (23.3 ± 7.5) were unaffected. There was no clinically apparent bleeding following administration of either anticoagulant. All shunts remained patent during the experiments. All devices initiated thrombosis in both untreated and treated animals. Fibrin deposition (mg) averaged 0.36 ± 0.19, 5.08 ± 0.35, and 2.66 ± 0.09 in untreated ePTFE, DG, and TG controls, respectively. These differences were likely related to differences in geometry, volume, texture, surface, and/or chemistry. The antithrombotic effects of heparin and aFXI in the DG or TG were comparable. Heparin reduced fibrin deposition to 1.58 ± 0.58 (31%; P < .001) and 1.01 ± 0.38 (38%; P < .02) in the DG and TG, respectively. Pretreatment with aFXI reduced fibrin deposition to 1.45 ± 0.45 (29%; P < .001) and 1.02 ± 0.55 (38%; P < .001) in the DG and TG, respectively. Thrombogenesis was minimal in the native ePTFE grafts with peak NPAR (billions)/(5-minute period) at (0.21 ± 0.18)/(60-55), compared with (1.49 ± 0.4)/(50-45) and (2.87 ± 0.46)/(40-35) in untreated DG and TG controls, respectively (Figure 1A-B). Heparin reduced NPAR between 10 to 60 minutes in DG and 15 to 60 minutes in the TG, with peak values reaching (0.38 ± 0.14)/(35-30) (26%; P < .02) and (0.30 ± 0.08)/(20-15) (10%; P < .01) in the 2 devices, respectively. Pretreatment with aFXI also reduced NPAR between 10 to 60 minutes in the DG and 20 to 60 minutes in the TG, with peak values of (0.44 ± 0.23)/(35-30) (30%; P < .05) and (0.587 ± 0.117)/(15-10) (20%; P > .05), respectively. Incorporated into untreated ePTFE, DG, and TG thrombi by 60 minutes were 1.57 ± 1.70, 10.78 ± 1.86, and 17.9 ± 2.01 billion platelets, respectively (Figure 1C-D). Following heparin, the DG and TG thrombi contained only 2.93 ± 0.62 (27%; P < .001) and 1.56 ± 0.56 (9%; P < .001) billion platelets, respectively. aFXI reduced the platelet contents of the DG and TG to 3.79 ± 1.2 (35%; P < .001) and 1.19 ± 1.11 (7%; P < .01) billion platelets, respectively. Antithrombotic effects of anticoagulants, such as heparin and aFXI, could result from reduced fibrin formation and platelet activation and/or from enhanced thrombolysis and/or embolization. FXI, unlike coagulation factors of the extrinsic and common pathways, is not essential for hemostasis.7 However, plasma FXI activity is required to occlusion following transvascular FeCl3-induced arterial injury and thrombosis in mice as well as the early postnatal mortality observed in protein C–deficient mice.8,9 Moreover, FXI is an independent risk factor for thrombosis in humans.10,11 About 0.5% of Ashkenazi Jews exhibit severe FXI deficiency.12 Interestingly, medical history of acute myocardial infarction (AMI) in such patients corresponds to the incidence of AMI in a matched general population.13 Since AMI has 30% to 50% mortality,14 it remains unclear whether FXI deficiency modifies the outcome of AMI. In our model, anticoagulation did not prevent initiation of thrombogenesis, but it markedly reduced the extent of thrombus propagation over time irrespective of the initiating trigger or the anticoagulant used. Thus, while FXI inhibition may not reduce the frequency of initial thrombotic events, thrombus growth rate, overall thrombus mass, and thrombo-occlusion may be reduced, thereby leading to more favorable outcomes. Accordingly, FXI appears to be a rational target for antithrombotic therapy. View larger version (36K): [in this window] [in a new window]   Figure 1.. Platelet accumulation onto thrombogenic devices. Platelet (PLT) accumulation was measured during the first 60 minutes of thrombus growth from the time of temporary DG, TG, or ePTFE (not displayed) device deployment at time 0 minute into chronic AV shunts. indicates untreated DG or TG controls; , heparin treatment; and , aFXI treatment. (A) Net PLT accumulation rates (NPAR) in the DG. (B) NPAR in the TF-dependent TG. (C) Net PLT accumulation in the DG. (D) Net PLT accumulation in the TG. Error bars correspond to 1 SD. Refer to "Results and discussion" for summary results on the limited thrombogenicity of native ePTFE.      Acknowledgements   We gratefully acknowledge Dr Hugh Hoogendoorn for the generous gift of the aFXI antibody, Dr Jim Morrissey for providing relipidated recombinant human tissue factor, and Dr Uri Seligsohn for his advice and sharing of data obtained in FXI deficient patients. We thank Tammy Hails and Ulla Marzec for their expert technical assistance.    Footnotes   Submitted January 31, 2003; accepted April 1, 2003. Prepublished online as Blood First Edition Paper, April 10, 2003; DOI 10.1182/blood-2003-01-0324. Supported by National Institutes of Health grants HL31469 (S.R.H.) and RR00165 (Yerkes National Primate Research Center), and by a grant from the National Science Foundation (EEC-9731643) to the Georgia Institute of Technology. This work has been presented in part at the 43rd and 44th annual meetings of the American Society of Hematology, 2001 and 2002, respectively. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: András Gruber, W. H. Coulter Department of Biomedical Engineering, Emory University School of Medicine, 1639 Pierce Dr, WMB Suite 2001, Atlanta, GA 30322-4600; e-mail: agruber{at}emory.edu.    References Top Abstract Introduction Study design Results and discussion References   Walsh PN. Roles of platelets and FXI in the initiation of blood coagulation by thrombin. Thromb Haemost. 2001;86: 75-82.[Medline] [Order article via Infotrieve] Lee A, Agnelli G, Buller H, et al. Dose-response study of recombinant factor VIIa/tissue factor inhibitor recombinant nematode anticoagulant protein c2 in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement. Circulation. 2001;104: 74-78.[Abstract/Free Full Text] Hanson SR, Griffin JH, Harker LA, Kelly AB, Esmon CT, Gruber A. Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates. J Clin Invest. 1993;92: 2003-2012.[Medline] [Order article via Infotrieve] Schneider PA, Kotzé HF, Heyns AD, Hanson SR. Thromboembolic potential of synthetic vascular grafts in baboons. J Vasc Surg. 1989;10: 75-82.[CrossRef][Medline] [Order article via Infotrieve] Despotis GJ, Joist JH, Hogue CW Jr, et al. The impact of heparin concentration and activated clotting time monitoring on blood conservation: a prospective, randomized evaluation in patients undergoing cardiac operation. J Thorac Cardiovasc Surg. 1995;110: 46-54.[Abstract/Free Full Text] Faraday N, Guallar E, Sera VA, et al. Utility of whole blood hemostatometry using the clot signature analyzer for assessment of hemostasis in cardiac surgery. Anesthesiology. 2002;96: 1115-1122.[Medline] [Order article via Infotrieve] Bolton-Maggs PH. Factor XI deficiency and its management. Haemophilia. 2000;6(suppl1): 100-109. Rosen ED, Gailani D, Castellino FJ. FXI is essential for thrombus formation following FeCl3-induced injury of the carotid artery in the mouse [letter]. Thromb Haemost 2002;87: 774-776.[Medline] [Order article via Infotrieve] Chan JC, Ganopolsky JG, Cornelissen I, et al. The characterization of mice with a targeted combined deficiency of protein C and factor XI. Am J Pathol. 2001;158: 469-479.[Abstract/Free Full Text] Meijers JC, Tekelenburg WL, Bouma BN, Bertina RM, Rosendaal FR. High levels of coagulation factor XI as a risk factor for venous thrombosis. N Engl J Med. 2000;342: 696-701.[Abstract/Free Full Text] Berliner JI, Rybicki AC, Kaplan RC, Monrad ES, Freeman R, Billett HH. Elevated levels of Factor XI are associated with cardiovascular disease in women. Thromb Res. 2002;107: 55-60.[Medline] [Order article via Infotrieve] Seligsohn U, Peretz H. Molecular genetics aspects of factor XI deficiency and Glanzmann thrombasthenia. Haemostasis. 1994;24: 81-85.[Medline] [Order article via Infotrieve] Salomon O, Steinberg D, Dardik R, et al. Severe factor XI deficiency confers no protection against acute myocardial infarction [abstract]. Blood. 2001;98: 260a. Tunstall-Pedoe H, Morrison C, Woodward M, Fitzpatrick B, Watt G. Sex differences in myocardial infarction and coronary deaths in the Scottish MONICA population of Glasgow 1985 to 1991: presentation, diagnosis, treatment, and 28-day case fatality of 3991 events in men and 1551 events in women. Circulation. 1996;93: 1981-1992.[Abstract/Free Full Text] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: T. C. White-Adams, M. A. Berny, E. I. Tucker, J. M. Gertz, D. Gailani, R. T. Urbanus, P. G. de Groot, A. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2003-01-0324v1 102/3/953    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gruber, A. Articles by Hanson, S. R. Search for Related Content PubMed PubMed Citation Articles by Gruber, A. Articles by Hanson, S. R. Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Brief Reports Social Bookmarking                   What's this?

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