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Fibrinolysis Profiles and Platelet Activation After Endothelial Cell Seeding of Prosthetic Vascular Grafts
Fibrinolysis Profiles and Platelet Activation After Endothelial Cell Seeding of Prosthetic Vascular Grafts J.V. Smyth, FRCS, M. Welch, FRCS, H.M.H. Carr, FRCS, P.D.F. Dodd, FRCS, P.R. Eisenberg, MD, and M.G. Walker ChM, Manchester, England, and St. Louis, Missouri
There is no convincing evidence that endothelial cell seeding of prosthetic grafts in humans confers any of the advantages seen in animals. However, partial endothelial coverage might exert a subtle effect not detectable with indirect end points such as patency or scintigraphy. This study examined seeded cell function by measuring fibrinolytic and platelet activation markers in patients receiving seeded and control prosthetic grafts. Thirty-two patients were randomly assigned to seeded (n = 15) and control (n = 17) groups. Preoperatively and 3, 6, and 12 months postoperatively, plasma levels of fibrinopeptide A (FPA), B1~1-42 fragment, cross-linked fibrin degradation products (XL-FDP), thromboxane A2 (TXA2), platelet factor 4 (PF4), and 13-thromboglobulin (13TG) were measured. Patients with seeded grafts had significantly lower levels of FPA at 6 and 12 months (p <0.05) and a significant overall group effect (p <0.05). These patients also tended to have higher levels of XL-FDP (p <0.1). No other significant differences were seen. The lower rate of conversion of fibrinogen to fibrin and the trend toward increased fibrinolysis seen in seeded grafts may be due to the metabolic effects of viable retained seeded cells. Although comparable platelet activation indicates that endothelial coverage remains limited, seeding may exert an antithrombotic influence at the graft surface. (Ann Vasc Surg 1995;9:542-546.)
Graft patency reflects the influence of m a n y factors such as smoking, medication compliance, site of distal anastomosis, flow dynamics, and graft diameter and type. Autologous vein carries primary patency rates of 84% at 3 years for femoropopliteal bypass overall; prosthetic grafts compare poorly with 35% patency) Clinical studies using patency or indirect assessment of the From the Department of Vascular Surgery, Manchester Royal Infirmary, Manchester, England, and Washington University School of Medicine (P.R.E.), Barnes Hospital, St. Louis, Mb. Supported in part by W.L. Gore & Associates, Inc., Flagstaff, Ariz. Presented at the Third International Meeting of the British/Swedkh Angiology Societies, Lund, Sweden, May 1994. Reprint requests: M.G. Walker, Consultant Vascular Surgeon, Department of Vascular Surgery, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL. 542
thrombogenicity of the flow surface have not confirmed the theoretic advantages of seeding in humans, although the validity of the seeding technique has been clearly demonstrated. 2 However, patients whose seeded grafts develop an incomplete endothelial lining might show differences in fibrinolytic profile and platelet activation products, and this study was designed to investigate this. MATERIAL AND METHODS Thirty-two patients between the ages of 41 and 75 years, who required prosthetic femoroproxima] poplitea] bypass for claudication in the absence of suitable vein and who would be expected to be available for at least a 1-year follow-up were recruited. Patients were randomly assigned to seeded (n = 15) and control (n = 17) groups.
Vol, 9, No. 6 1995
Patients requiring "redo," combined, or emergency procedures and those who had undergone angioplasty within the past 30 days were excluded, as were those with abnormal biochemistry, hematology, or coagulation profiles. The study was approved by the ethics committee and informed consent was obtained from all patients prior to surgery. Preoperatively all patients underwent arteriography to document the extent of disease and vascular laboratory assessment. Complete operative details were recorded, and 6 m m thin-walled expanded polytetrafluoroethylene grafts (W.L. Gore & Associates, Inc., Flagstaff, Ariz.) were used in all patients. Grafts were preclotted by filling them with autologous unheparinized blood for 45 minutes, and then excess clot material was removed by gentle passage of a balloon embolectomy catheter. Endothelial cell seeding was performed using an endothelial cell harvesting kit (W.L. Gore & Associates, Inc.) and the seeded cells were incubated in the preclotted graft for 20 minutes prior to implantation. Cell harvest was assessed by culture of aliquots of seeding solution to determine cell numbers and viability and by examination of donor vein segments by scanning electron microscopy to confirm endothelial denudation. Preclotted control grafts were sham seeded with Hanks' balanced salt solution. In the postoperative period all patients received aspirin, 300 mg/day, and dipyridamole, 100 mg three times/day, and were followed up in the vascular clinic by clinical examination and vascular laboratory assessment. Preoperatively and at 3, 6, and 12 months postoperatively, blood was drawn from each patient, regardless of graft patency, using an atraumatic, sterile double-syringe technique and placed into anticoagulant tubes in ice. Plasma specimens were prepared by centrifugation at 1500 g for 15 minutes and then frozen at - 7 0 ~ C until assay. Samples were assayed for markers of platelet activation and fibrinolysis (Table I). All assays were performed by experienced laboTable I. Assays performed Fibrinolytic Markers Fibrinopeptide A B[31-42 Thromboxane A2 Platelet Activation Markers Platelet factor 4 [3-Thromboglobulin Cross-linked fibrin degradation products
Fibrinolysis, platelets, and seeded prosthetic grafts
543
ratory technicians. A radioimmunoassay technique using polyclonal antiserum (Byck-Sangtek, Deitzenbach, Germany) 3 was used for fibrinopeptide A (FPA) and a radioimmunoassay based on tritiated tracer (Dupont Medical Products/New England Nuclear, Boston, Mass. ) and lupine antibody (Upjohn Co., Kalamazoo, Mich.) for thromboxane B 2 ( T X B 2 ) . 4 Commercially available radioimmunoassay kits for [3-thromboglobulin (BTG) (Amersham Corp., Arlington Heights, Ill.), platelet factor 4 (PF4) (Abbott Laboratories, Abbott Park, Ill.), and B[31-42 fragment were used. Cross-linked fibrin degradation products (XL-FDP) were measured by a monoclonal antibody-based enzymelinked immunosorbent assay (Dimer-Test, American Diagnostica, Greenwich, Conn.).2 Results were interpreted by unpaired two-tailed t tests and analysis of variance (ANOVA) for measurements relating to graft treatment, with a commercial software program (Statview II, Abacus Concepts, Berkeley, Calif.) on a Macintosh computer. RESULTS Harvested cells were viable in culture and mean seeding densities of 1.1 • 104 cefis/cm2 (standard deviation 0.58 • 104) were obtained. Complete denudation of the donor vein segment was routinely observed. Two grafts from each group occluded during the course of the study, which was insufficient for meaningful analysis with regard to patency. There were no statistically significant differences in the platelet activation markers PF4, 6TG, or TXA2 between seeded and control groups at any time. B~31-42 levels were normal in all patients at all times. EPA levels were significantly lower (p < 0.05 ) in the seeded group at 6 and 12 months postoperatively. When all values were analyzed by ANOVA for repeated measures, the difference between the groups was significant (p = 0.03)(Fig. 1). Although the seeded group had consistently elevated values for XL-FDP, there were no significant differences at the 0.05 level at any single time point by unpaired t tests. Overall analysis with ANOVA for repeated measures indicated that the observed trend approached significance (p = 0.08) (fig. 2). DISCUSSION The results of seeding prosthetic grafts with endothelial cells in the clinical setting have been disappointing after promising h u m a n in vitro
544
Annals of Vascular Surgery
Smyth et aI.
15 D seeded 9
E
n u-
unseeded
10
--t
5
89 I
pre
3
6
12
time /months Fig. l. Plasma levels of fibrinopeptide A (FPA).
300
E
w
2OO
c
n t~ I.L
I 9-J x
100
0 )re
[]
seeded
m
unseeded
I
l
I
3
6
12
time /months Fig. 2. Plasma levels of cross-linked fibrin degradation products (XL-FDP).
Vol. 9, No. 6
1995
Fibrinolysis, platelets, and seeded prosthetic grafts
~ lasmin ~bri~gen
f~p
(TXB2) ~XA2~
ISTGi Fig. 3. Derivation of fibrinolysis (left) and platelet activation (right) products. B[31-42 = B[31-42 fibrinogen fragment; TXA2/TXB 2 = t h r o m b o x a n e A2/B2; XL-FDP = cross-linked fibrin degradation products; PF4 = platelet factor 4; FPA = fibrinopeptide A; [3TG= [3-thromboglobulin.
studies and a clear demonstration of the benefits in in vivo animal studies. 6 With the relatively low seeding densities that can be achieved by singlestage acute seeding from donor vein segments, a confluent monolayer cannot be achieved at the time of seeding. Subsequent development of endothelial confluence requires endothelial migration across the graft surface, and there is extensive evidence indicating that this does not happen in h u m a n s as it does animals. Successful seeding is expected to confer several advantages including resistance to infection7 and reduced neointimal hyperplasiafl but most important improved patency. 9'~~ An endothelial lining would contribute to this both by the passive presence of a layer of nonthrombogenic endothelial cells and by the active secretion by those cells of antithrombotic and fibrinolytic factors. The absence of a detectable monolayer does not preclude the presence of localized islands of viable seeded cells, which might fulfill this latter function. Studies examining platelet kinetics or deposition have shown that prosthetic grafts significantly reduce platelet survival time (PST) in humans T M and have a significantly greater thrombogenicity compared to the native vessel. T M Following seeding, no clear reduction in throm-
545
bogenicity or preservation of PST has been consistently demonstrated, ~-18 although only largescale changes can be reliably detected using existing techniques such as indium-labeled platelet scintigraphy. Increased platelet activation by prosthetic grafts can also be assessed by measuring the levels of their release products in plasma. ~9-2~ Activated platelets release a wide range of cytokines and cell mediators including platelet-specific and nonspecific proteins (Fig. 3). PF4 is a platelet-specific tetrameric protein/proteoglycan complex released from platelet or-granules on activation that rapidly binds to the endothelial surface where it can inactivate heparin directly. Based on antithrombin III binding studies, high- and low-affinity forms of PF4 have been identified, the latter of which is subsequently hydrolyzed to [3TG. Although several biologic activities of both PF4 and [3TG have been identified, their physiologic importance is still unknown, but they are an accurate measure of platelet secretion as platelet-poor plasma levels are negligible. TXA2 is a short-lived prostanoid with vasoconstrictor and platelet aggregation activity derived from arachidonic acid by the action of phospholipase and platelet cytoplasm cyclo-oxygenases on platelet activation. TXA2 is measured indirectly using serum levels of its stable metabolite TXB2, which have been shown experimentally to correlate with rates of platelet deposition. 22 In this study no difference was found in platelet activation, as demonstrated by PF4, [3TG, or TXA2, between control and seeded grafts at any time after implantation, both groups showing more platelet activation than normal despite antiplatelet therapy. These results are in agreement with those of previous studies where seeded and nonseeded grafts could not be differentiated using plasma markers up to a year after implantation. 1~'16 This implies that acute seeding either does not lead to the development of a confluent monolayer in the graft or alters the function of the seeded endothelial cells so that they are unable to prevent the activation of circulating platelets by the prosthesis. Although complete reendothelialization is associated with normalization of P S T ~ 23"25 the relationship between subconfluent coverage and recovery of PST remains unknown. The development of thrombosis is dependent on the balance between the thrombin-stimulated conversion of fibrinogen to fibrin and the subsequent degradation of the cross-linked fibrin lattices by plasmin (Fig. 3). FPA and B[31-42 are both fibrinogen cleavage fragments; FPA is re-
546
Smyth et al.
leased from the fibrinogen a-chain by thrombin and B[31-42 fragment from the [3-chain by plasmin. Taken together FPA and B[31-42 plasma levels reflect the rate of formation of fibrin from fibrinogen and therefore are valuable markers of ongoing thrombosis) 6'27 Levels of B[31-42 were not raised in our patients, which is consistent with previous data and suggests that plasma-mediated fibrinogen degradation is an infrequent consequence of increased physiologic fibrinolytic activity. The lower levels of FPA in these patients indicate that the rate of formation of fibrin from fibrinogen is significantly decreased and m a y reflect less procoagulant activity related to seeding of grafts. XL-FDP are produced by plasmin acting on fibrin lattices, which form a heterogeneous group of peptides containing D-dimer products and are sensitive and specific markers of fibrinolytic activity. The trend toward elevated levels of XL-FDP in seeded grafts in this study, although not significant in the small sample size, is suggestive of increased physiologic fibrinolytic activity in these patients. When considered with the lower levels of FPA in seeded patients, this implies a more favorable balance of fibrinolytic compared to procoagulant activity.
CONCLUSION Endothelial cell seeding may provide some protection against in situ thrombosis in grafts by the fibrinolytic activity of the surviving seeded cells and their progeny. The survival time of circulating platelets is unaffected, indicating that most of the graft surface remains exposed, and the presence of islands of functional endothelial coverage appears possible. REFERENCES 1. Rutherford RB, Jones DN, Bergentz SE, et al. Factors affecting the patency of infrainguinal bypass. J Vasc Surg 1988;8: 236-246. 2. T h o m s o n GJL, Vohra RK, Carr HMH, et al. Adult h u m a n endothelial cell seeding u s i n g expanded polytetrafuoroethylene vascular grafts: A comparison of four substrates. Surgery 1991;109:20-27. 3. Eisenberg PR, S h e r m a n LA, Schectman K, et al. Fibrinopeptide A: A m a r k e r of acute coronary thrombosis. Circulation 1985;71:912-918. 4. Fitzpatrick FA, G o r m a n RR, McGuire JC, et al. A radioimmun o a s s a y for t h r o m b o x a n e B2. Anal Biochem 1977;82:1-7. 5. Rylatt DB, Blake AS, Cattis DA, et al. An i m m u n o a s s a y for h u m a n D-dimer u s i n g monoclonal antibodies. Thromb Res 1983;31:767-778. 6. Welch M, Durrans D, Carr HMH, et al. Endothelial cell seeding: A review. A n n Vasc Surg 1992;6:473-484. 7. Birinyi LK, Douville EC, Lewis SA, et aL Increased resistance to bacteremic graft infection after endothelial cell seeding. J Vasc Surg 1987;5:193-197.
Annals of Vascular Surgery
8. Bush HL, Jakubowski JA, Sentissi JA, et al. Neointimal hyperplasia occurring after carotid endarterectomy in a canine model: Effect of endothelial cell seeding vs. perioperative aspirin. J Vasc Surg 1987;5:118-125. 9. Stanley JC, Burkel WE, Ford JW, et al. Enhanced patency of small diameter, externally supported Dacron iliofemoral grafts seeded with endothelial ceils. Surgery 1982;92:9941005. 10. Allen BT, Long JA, Clark RE, et al. Influence of endothelial cell seeding on platelet deposition and patency in small diameter Dacron arterial grafts. J Vasc Surg 1984;1:224-233. i1. McCollum CN, Kester R, Rajah SM, et al. Arterial graft maturation: The duration of thrombotic activity in Dacron aortobifemoral grafts m e a s u r e d by platelet and fibrinogen kinetics. Br J Surg 198i;68:61-64. 12. G o l d m a n M, Norcott HC, Hawker RJ, et al. Platelet a c c u m u lation on m a t u r e Dacron grafts in m a n . Br J Surg 1982;69: $38-$40. 13. Stratton JR, Thiele BL, Ritchie JL. Platelet deposition on Dacron aortic bifurcation grafts in m a n : Qnantitation with indium- 111 platelet imaging. Circulation I982;66:1287-1293. 14. Callow AD, Connolly R, O'Donnell TE et al. Platelet-arterial synthetic graft interaction and its modification. Arch Surg 1982;117:1447-1455. 15. Zilla P, Fasol R, Deutsch M, et al. Endothelial cell seeding of polytetrafluoroethylene vascular grafts in h u m a n s : A preliminary report. J Vasc Surg 1987;6:535-541. 16. Fasol R, Zilla P, Deutsch M, et al. H u m a n endothelial cell seeding: Evaluation of its effectiveness by platelet parameters after one year. J Vasc Surg 1989;9:432-436. 17. Ortenwall P, Wadenvik H, Kutti J, et al. Endothelial cell seeding reduces thrombogenicity of Dacron grafts in h u m a n s . J Vasc Surg 1990;11:403-410. 18. Ortenwall P, Wadenvik I-I, Risberg B. Reduced platelet deposition on seeded versus unseeded s e g m e n t s of expanded polytetrafluoroethylene grafts: Clinical observations after a 6 - m o n t h follow-up. J Vasc Surg 1989;10:374-380. 19. W a k e f e l d TW, Shulkin BL, Fellows EP, et al. Platelet reactivity in h u m a n aortic grafts: A prospective, randomized midterm study of platelet adherence and release products in Dacron and polytetrafluoroethylene conduits. J Vasc Surg 1989;9:234-243. 20. Walz DA. Platelet-released proteins as molecular markers for t h e activation process. Semin Thromb Hemost 1984;10:270279. 2i. Messmore HL, Walenga JM, Fareed J. Molecular markers of platelet activation. Semin Thromb Hemost 1984;10:264-269. 22. Wakefield TW, Lindblad B, G r a h a m LM, et al. Nuclide imaging of vascular graft-platelet interactions: Comparison of i n d i u m excess a n d t e c h n e t i u m subtraction techniques. J Surg Res 1986;40:388-394. 23. Claggett GP, Burkel WE, Sharefkin JB, et al. Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells. Circulation 1984;69:632-636. 24. Sharefkin JB, Latker C, Smith M, et al. Early normalization of platelet survival by endothelial cell seeding of Dacron arterial prostheses in dogs. Surgery i982;92:385-393. 25. Harker LA, Slichter SJ, Sauvage LR. Platelet c o n s u m p t i o n by arterial prostheses: The effects of endothelialization and pharmacological inhibition of platelet function. A n n Surg 1977;186:594-601. 26. Warenga JM, Fareed J, Mariani G, et al. Diagnostic efficacy of a simple r a d i o i m m u n o a s s a y t e s t for fibrinogen/fibrin fragm e n t s containing the B[315-42 sequence. Semin Thromb Hemost 1984;10:252-263. 27. Warenga JM, Hoppensteadt D, Emanuele RM, et al. Perform a n c e characteristics of a simple r a d i o i m m u n o a s s a y test for fibrinopeptide A. Semin Thromb Hemost 1984;10:219-227.
There is no convincing evidence that endothelial cell seeding of prosthetic grafts in humans confers any of the advantages seen in animals. However, partial endothelial coverage might exert a subtle effect not detectable with indirect end points such as patency or scintigraphy. This study examined seeded cell function by measuring fibrinolytic and platelet activation markers in patients receiving seeded and control prosthetic grafts. Thirty-two patients were randomly assigned to seeded (n = 15) and control (n = 17) groups. Preoperatively and 3, 6, and 12 months postoperatively, plasma levels of fibrinopeptide A (FPA), B1~1-42 fragment, cross-linked fibrin degradation products (XL-FDP), thromboxane A2 (TXA2), platelet factor 4 (PF4), and 13-thromboglobulin (13TG) were measured. Patients with seeded grafts had significantly lower levels of FPA at 6 and 12 months (p <0.05) and a significant overall group effect (p <0.05). These patients also tended to have higher levels of XL-FDP (p <0.1). No other significant differences were seen. The lower rate of conversion of fibrinogen to fibrin and the trend toward increased fibrinolysis seen in seeded grafts may be due to the metabolic effects of viable retained seeded cells. Although comparable platelet activation indicates that endothelial coverage remains limited, seeding may exert an antithrombotic influence at the graft surface. (Ann Vasc Surg 1995;9:542-546.)
Graft patency reflects the influence of m a n y factors such as smoking, medication compliance, site of distal anastomosis, flow dynamics, and graft diameter and type. Autologous vein carries primary patency rates of 84% at 3 years for femoropopliteal bypass overall; prosthetic grafts compare poorly with 35% patency) Clinical studies using patency or indirect assessment of the From the Department of Vascular Surgery, Manchester Royal Infirmary, Manchester, England, and Washington University School of Medicine (P.R.E.), Barnes Hospital, St. Louis, Mb. Supported in part by W.L. Gore & Associates, Inc., Flagstaff, Ariz. Presented at the Third International Meeting of the British/Swedkh Angiology Societies, Lund, Sweden, May 1994. Reprint requests: M.G. Walker, Consultant Vascular Surgeon, Department of Vascular Surgery, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL. 542
thrombogenicity of the flow surface have not confirmed the theoretic advantages of seeding in humans, although the validity of the seeding technique has been clearly demonstrated. 2 However, patients whose seeded grafts develop an incomplete endothelial lining might show differences in fibrinolytic profile and platelet activation products, and this study was designed to investigate this. MATERIAL AND METHODS Thirty-two patients between the ages of 41 and 75 years, who required prosthetic femoroproxima] poplitea] bypass for claudication in the absence of suitable vein and who would be expected to be available for at least a 1-year follow-up were recruited. Patients were randomly assigned to seeded (n = 15) and control (n = 17) groups.
Vol, 9, No. 6 1995
Patients requiring "redo," combined, or emergency procedures and those who had undergone angioplasty within the past 30 days were excluded, as were those with abnormal biochemistry, hematology, or coagulation profiles. The study was approved by the ethics committee and informed consent was obtained from all patients prior to surgery. Preoperatively all patients underwent arteriography to document the extent of disease and vascular laboratory assessment. Complete operative details were recorded, and 6 m m thin-walled expanded polytetrafluoroethylene grafts (W.L. Gore & Associates, Inc., Flagstaff, Ariz.) were used in all patients. Grafts were preclotted by filling them with autologous unheparinized blood for 45 minutes, and then excess clot material was removed by gentle passage of a balloon embolectomy catheter. Endothelial cell seeding was performed using an endothelial cell harvesting kit (W.L. Gore & Associates, Inc.) and the seeded cells were incubated in the preclotted graft for 20 minutes prior to implantation. Cell harvest was assessed by culture of aliquots of seeding solution to determine cell numbers and viability and by examination of donor vein segments by scanning electron microscopy to confirm endothelial denudation. Preclotted control grafts were sham seeded with Hanks' balanced salt solution. In the postoperative period all patients received aspirin, 300 mg/day, and dipyridamole, 100 mg three times/day, and were followed up in the vascular clinic by clinical examination and vascular laboratory assessment. Preoperatively and at 3, 6, and 12 months postoperatively, blood was drawn from each patient, regardless of graft patency, using an atraumatic, sterile double-syringe technique and placed into anticoagulant tubes in ice. Plasma specimens were prepared by centrifugation at 1500 g for 15 minutes and then frozen at - 7 0 ~ C until assay. Samples were assayed for markers of platelet activation and fibrinolysis (Table I). All assays were performed by experienced laboTable I. Assays performed Fibrinolytic Markers Fibrinopeptide A B[31-42 Thromboxane A2 Platelet Activation Markers Platelet factor 4 [3-Thromboglobulin Cross-linked fibrin degradation products
Fibrinolysis, platelets, and seeded prosthetic grafts
543
ratory technicians. A radioimmunoassay technique using polyclonal antiserum (Byck-Sangtek, Deitzenbach, Germany) 3 was used for fibrinopeptide A (FPA) and a radioimmunoassay based on tritiated tracer (Dupont Medical Products/New England Nuclear, Boston, Mass. ) and lupine antibody (Upjohn Co., Kalamazoo, Mich.) for thromboxane B 2 ( T X B 2 ) . 4 Commercially available radioimmunoassay kits for [3-thromboglobulin (BTG) (Amersham Corp., Arlington Heights, Ill.), platelet factor 4 (PF4) (Abbott Laboratories, Abbott Park, Ill.), and B[31-42 fragment were used. Cross-linked fibrin degradation products (XL-FDP) were measured by a monoclonal antibody-based enzymelinked immunosorbent assay (Dimer-Test, American Diagnostica, Greenwich, Conn.).2 Results were interpreted by unpaired two-tailed t tests and analysis of variance (ANOVA) for measurements relating to graft treatment, with a commercial software program (Statview II, Abacus Concepts, Berkeley, Calif.) on a Macintosh computer. RESULTS Harvested cells were viable in culture and mean seeding densities of 1.1 • 104 cefis/cm2 (standard deviation 0.58 • 104) were obtained. Complete denudation of the donor vein segment was routinely observed. Two grafts from each group occluded during the course of the study, which was insufficient for meaningful analysis with regard to patency. There were no statistically significant differences in the platelet activation markers PF4, 6TG, or TXA2 between seeded and control groups at any time. B~31-42 levels were normal in all patients at all times. EPA levels were significantly lower (p < 0.05 ) in the seeded group at 6 and 12 months postoperatively. When all values were analyzed by ANOVA for repeated measures, the difference between the groups was significant (p = 0.03)(Fig. 1). Although the seeded group had consistently elevated values for XL-FDP, there were no significant differences at the 0.05 level at any single time point by unpaired t tests. Overall analysis with ANOVA for repeated measures indicated that the observed trend approached significance (p = 0.08) (fig. 2). DISCUSSION The results of seeding prosthetic grafts with endothelial cells in the clinical setting have been disappointing after promising h u m a n in vitro
544
Annals of Vascular Surgery
Smyth et aI.
15 D seeded 9
E
n u-
unseeded
10
--t
5
89 I
pre
3
6
12
time /months Fig. l. Plasma levels of fibrinopeptide A (FPA).
300
E
w
2OO
c
n t~ I.L
I 9-J x
100
0 )re
[]
seeded
m
unseeded
I
l
I
3
6
12
time /months Fig. 2. Plasma levels of cross-linked fibrin degradation products (XL-FDP).
Vol. 9, No. 6
1995
Fibrinolysis, platelets, and seeded prosthetic grafts
~ lasmin ~bri~gen
f~p
(TXB2) ~XA2~
ISTGi Fig. 3. Derivation of fibrinolysis (left) and platelet activation (right) products. B[31-42 = B[31-42 fibrinogen fragment; TXA2/TXB 2 = t h r o m b o x a n e A2/B2; XL-FDP = cross-linked fibrin degradation products; PF4 = platelet factor 4; FPA = fibrinopeptide A; [3TG= [3-thromboglobulin.
studies and a clear demonstration of the benefits in in vivo animal studies. 6 With the relatively low seeding densities that can be achieved by singlestage acute seeding from donor vein segments, a confluent monolayer cannot be achieved at the time of seeding. Subsequent development of endothelial confluence requires endothelial migration across the graft surface, and there is extensive evidence indicating that this does not happen in h u m a n s as it does animals. Successful seeding is expected to confer several advantages including resistance to infection7 and reduced neointimal hyperplasiafl but most important improved patency. 9'~~ An endothelial lining would contribute to this both by the passive presence of a layer of nonthrombogenic endothelial cells and by the active secretion by those cells of antithrombotic and fibrinolytic factors. The absence of a detectable monolayer does not preclude the presence of localized islands of viable seeded cells, which might fulfill this latter function. Studies examining platelet kinetics or deposition have shown that prosthetic grafts significantly reduce platelet survival time (PST) in humans T M and have a significantly greater thrombogenicity compared to the native vessel. T M Following seeding, no clear reduction in throm-
545
bogenicity or preservation of PST has been consistently demonstrated, ~-18 although only largescale changes can be reliably detected using existing techniques such as indium-labeled platelet scintigraphy. Increased platelet activation by prosthetic grafts can also be assessed by measuring the levels of their release products in plasma. ~9-2~ Activated platelets release a wide range of cytokines and cell mediators including platelet-specific and nonspecific proteins (Fig. 3). PF4 is a platelet-specific tetrameric protein/proteoglycan complex released from platelet or-granules on activation that rapidly binds to the endothelial surface where it can inactivate heparin directly. Based on antithrombin III binding studies, high- and low-affinity forms of PF4 have been identified, the latter of which is subsequently hydrolyzed to [3TG. Although several biologic activities of both PF4 and [3TG have been identified, their physiologic importance is still unknown, but they are an accurate measure of platelet secretion as platelet-poor plasma levels are negligible. TXA2 is a short-lived prostanoid with vasoconstrictor and platelet aggregation activity derived from arachidonic acid by the action of phospholipase and platelet cytoplasm cyclo-oxygenases on platelet activation. TXA2 is measured indirectly using serum levels of its stable metabolite TXB2, which have been shown experimentally to correlate with rates of platelet deposition. 22 In this study no difference was found in platelet activation, as demonstrated by PF4, [3TG, or TXA2, between control and seeded grafts at any time after implantation, both groups showing more platelet activation than normal despite antiplatelet therapy. These results are in agreement with those of previous studies where seeded and nonseeded grafts could not be differentiated using plasma markers up to a year after implantation. 1~'16 This implies that acute seeding either does not lead to the development of a confluent monolayer in the graft or alters the function of the seeded endothelial cells so that they are unable to prevent the activation of circulating platelets by the prosthesis. Although complete reendothelialization is associated with normalization of P S T ~ 23"25 the relationship between subconfluent coverage and recovery of PST remains unknown. The development of thrombosis is dependent on the balance between the thrombin-stimulated conversion of fibrinogen to fibrin and the subsequent degradation of the cross-linked fibrin lattices by plasmin (Fig. 3). FPA and B[31-42 are both fibrinogen cleavage fragments; FPA is re-
546
Smyth et al.
leased from the fibrinogen a-chain by thrombin and B[31-42 fragment from the [3-chain by plasmin. Taken together FPA and B[31-42 plasma levels reflect the rate of formation of fibrin from fibrinogen and therefore are valuable markers of ongoing thrombosis) 6'27 Levels of B[31-42 were not raised in our patients, which is consistent with previous data and suggests that plasma-mediated fibrinogen degradation is an infrequent consequence of increased physiologic fibrinolytic activity. The lower levels of FPA in these patients indicate that the rate of formation of fibrin from fibrinogen is significantly decreased and m a y reflect less procoagulant activity related to seeding of grafts. XL-FDP are produced by plasmin acting on fibrin lattices, which form a heterogeneous group of peptides containing D-dimer products and are sensitive and specific markers of fibrinolytic activity. The trend toward elevated levels of XL-FDP in seeded grafts in this study, although not significant in the small sample size, is suggestive of increased physiologic fibrinolytic activity in these patients. When considered with the lower levels of FPA in seeded patients, this implies a more favorable balance of fibrinolytic compared to procoagulant activity.
CONCLUSION Endothelial cell seeding may provide some protection against in situ thrombosis in grafts by the fibrinolytic activity of the surviving seeded cells and their progeny. The survival time of circulating platelets is unaffected, indicating that most of the graft surface remains exposed, and the presence of islands of functional endothelial coverage appears possible. REFERENCES 1. Rutherford RB, Jones DN, Bergentz SE, et al. Factors affecting the patency of infrainguinal bypass. J Vasc Surg 1988;8: 236-246. 2. T h o m s o n GJL, Vohra RK, Carr HMH, et al. Adult h u m a n endothelial cell seeding u s i n g expanded polytetrafuoroethylene vascular grafts: A comparison of four substrates. Surgery 1991;109:20-27. 3. Eisenberg PR, S h e r m a n LA, Schectman K, et al. Fibrinopeptide A: A m a r k e r of acute coronary thrombosis. Circulation 1985;71:912-918. 4. Fitzpatrick FA, G o r m a n RR, McGuire JC, et al. A radioimmun o a s s a y for t h r o m b o x a n e B2. Anal Biochem 1977;82:1-7. 5. Rylatt DB, Blake AS, Cattis DA, et al. An i m m u n o a s s a y for h u m a n D-dimer u s i n g monoclonal antibodies. Thromb Res 1983;31:767-778. 6. Welch M, Durrans D, Carr HMH, et al. Endothelial cell seeding: A review. A n n Vasc Surg 1992;6:473-484. 7. Birinyi LK, Douville EC, Lewis SA, et aL Increased resistance to bacteremic graft infection after endothelial cell seeding. J Vasc Surg 1987;5:193-197.
Annals of Vascular Surgery
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