Saratin, an inhibitor of von Willebrand factor–dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model☆☆☆★
Article Outline
Abstract
Purpose: Post–carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis , which is thought to act by binding to collagen, and inhibits von Willebrand factor–collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application. Methods: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area. Results: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 ± 17 vs 155 ± 33 platelets per grid, P = .05), and 24 hours after carotid endarterectomy (35 ± 11 vs 149 ± 37 platelets per grid, P = .0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% ± 1.8% vs 29.8% ± 6.8%, P = .0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P = .0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy. Conclusion: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation. (J Vasc Surg 2001;34:724-9.)
Carotid endarterectomy may be complicated by the development of thrombosis or intimal hyperplasia. Clinical studies demonstrate a post–carotid endarterectomy stroke rate of 1% to 10% in the immediate postoperative period,1 mostly due to thrombosis, and a restenosis rate of 10% to 20% at 2 to 5 years postoperatively, most of which is due to intimal hyperplasia.2 Thrombosis develops when platelets bind to the exposed subendothelial collagen by means of the von Willebrand factor and become activated, thereby enhancing adhesion to other platelets and encouraging thrombus formation. Intimal hyperplasia development is initiated by the accumulation of a monolayer of platelets. The adherence of platelets to subendothelial collagen leads to their activation, their degranulation, and the release of mitogenic and chemotactic factors, which contribute to smooth muscle cell activation followed by the development of intimal hyperplasia. In this study, we hypothesize that local interference with the development of platelet adhesion to an exposed collagen surface will decrease the subsequent development of thrombosis and intimal hyperplasia after a rat carotid endarterectomy.
A rat carotid endarterectomy model that involved an arteriotomy and direct removal of the intima/media layer previously described by our group3 was used. Saratin, a novel agent that has been suggested to inhibit platelet to collagen adhesion through inhibition of von Willebrand factor binding to collagen and that can be used in a site-specific manner by way of topical administration directly onto the endarterectomized surface, was used in this model.
Saratin is a 12,000-d recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis and is thought to block the adhesion of platelets to collagen. Saratin's proposed ability to affect the local environment of an endarterectomized vessel without requiring systemic distribution and without altering platelet function or coagulation makes it ideal for topical application during vascular surgical procedures.
Materials and methods
Animals
Sprague-Dawley rats (350-400 g) were organized into carotid endarterectomy groups on the basis of two main objectives: (1) evaluation of platelet adhesion and (2) evaluation of lumen stenosis due to intimal hyperplasia, as well as thrombosis rate. Within these two objectives, rats were divided into control and saratin-treated animals. All rats underwent a carotid endarterectomy; saratin-treated rats received a topical application of 5-μL solution of saratin on the luminal surface of the carotid artery immediately after endarterectomy. The platelet-adhesion group was evaluated by means of electron microscopy at 3 hours after carotid endarterectomy (n = 17) and 24 hours after carotid endarterectomy (n = 19). The intimal hyperplasia and thrombosis groups (n = 25) were harvested 2 weeks after carotid endarterectomy.
Carotid endarterectomy operation
The operative procedure has been previously described by our group3: rats were anesthetized, and the right common carotid artery was exposed. Arterial control was obtained and an arteriotomy performed. The intima and media were removed with microforceps, and the arteriotomy was closed.
Platelet-adhesion group
In the platelet-adhesion subgroups, the rats were reanesthetized, and the endarterectomized carotid arteries were harvested and fixed in a 4% glutaldehyde solution at 3 hours or 24 hours after carotid endarterectomy. The arteries were postfixed with osmium tetroxide, dehydrated in a graded alcohol series, dried by critical point with carbon dioxide (1072 psi and 31.1°C), coated with gold palladium, and placed in the scanning electron microscope (JEOL JSM 5410; JEOL, USA, Peabody, Mass). The endarterectomized areas were scanned at 2000× magnification and photographed. A collage of photographs was assembled and covered with a transparent overlay grid that consisted of 116 squares that were used to count the total number of platelets in each photograph. Platelet counts were performed by two blinded observers.
Intimal hyperplasia group
At 2 weeks after carotid endarterectomy, rats in the intimal hyperplasia and thrombosis groups were anesthetized, and the endarterectomized carotid artery was exposed. The vena cava was transected and the distal aorta cannulated with a 20-gauge catheter to infuse normal saline at 100 mm Hg until the vena cava effluent ran clear. Next, 10% buffered formalin was infused at 100 mm Hg in an equal volume to complete the perfusion-fixation technique. A 1-cm section of the operated carotid artery was dissected and placed in 10% formalin. The arteries were blocked with paraffin, sectioned, and elastin stained with Verhoeff's and van Gieson's stain. Multiple sections were taken at intervals of 3 μm each continuing along the distance of the continuous 10-0 nylon suture arteriotomy closure to standardize the region of sectioning. The elastin-stained slides were photographed with a Kodak DC 120 Zoom digital camera (Eastman Kodak Company, Rochester, NY). Any thrombosed sections were noted at this time. Nonthrombosed images were downloaded into a computer, and the luminal areas of the carotids were analyzed with the National Institutes of Health (Bethesda, Md) ImageJ Software program, Version 0.99i. This software package allowed us to delineate the inner area of the intimal hyperplasia and thus obtain an accurate measure of the cross-sectional area of the vessel lumen. The characteristic pattern of intimal hyperplasia was used to identify the demarcation between the outer limits of intimal hyperplasia and the medial/adventitial layers. The difference between the two areas (outer area of intimal hyperplasia minus the actual lumen) was determined as the absolute area of intimal hyperplasia. Because the arterial cross section had individual variations of shape, the values were expressed as a ratio of the absolute area of intimal hyperplasia to the outer limit of intimal hyperplasia and was reported as a percent lumen stenosis. This ratio represents the proportion of the lumen area occupied by intimal hyperplasia and allowed for comparison of the arterial cross sections of varying size.4 Minimal variability was seen between measurements with two blinded observers.
Bleeding times and platelet counts
Platelet counts and bleeding times of 12 rats were determined. All rats had platelet counts and bleeding times preoperatively. Six rats underwent a carotid endarterectomy and were harvested 3 hours postoperatively with bleeding times and platelet counts measured, and the remaining six rats were harvested 24 hours postoperatively with bleeding times and platelet counts measured. Three of the rats in each time group received topical saratin, and the remaining three rats served as controls.
Bleeding times were determined by transecting the distal 2 mm of the rat's tail and submerging 4 cm of the tail into a solution of phosphate-buffered solution at 37°C. The amount of time that elapsed from tail transection to cessation of bleeding was measured and was assigned as the bleeding time. Platelet counts were determined by drawing a 1- to 1.5-cc sample of blood from the internal jugular vein, which was analyzed in a Coulter STKS blood analyzer (Brea, Calif), and the results were expressed ×103. For the assessment of saratin's effect on platelet counts, the difference in platelet counts was determined for each rat by subtracting the preoperative platelet count from the postoperative platelet count to obtain a difference score.
This study was approved by the Institutional Review Board. All animal care complied with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Washington: National Academy Press, 1996.
Statistical methods
Please see the Appendix (online only) for details about statistical methods.
Results
The topical application of saratin onto exposed subendothelium after carotid endarterectomy significantly decreases the number of adherent platelets. The antiplatelet adherence effect of saratin was evaluated after two different postoperative times, 3 hours and 24 hours. The difference in the number of adherent platelets at 3 hours (Fig 1) and 24 hours (Fig 2) after carotid endarterectomy was significantly reduced in the rats treated with saratin as compared with the control rats.
Fig. 1.
Number of platelets adherent to exposed subendothelial surface 3 hours after carotid endarterectomy, comparing saratin-treated group (n = 7) and control group (n = 10). Data are means ± SE. Platelets were counted with scanning electron microscope. Saratin group received 5 μL of saratin solution applied topically to exposed subendothelial surface. Star indicates P value of .05.
Fig. 2.
Number of platelets adherent to exposed subendothelial surface 24 hours after carotid endarterectomy comparing saratin-treated group (n = 9) and control group (n = 10). Data are means ± SE. Platelets were counted with scanning electron microscope. Saratin group received 5 μL of saratin solution applied topically to exposed subendothelial surface. Star indicates P value of .01.
Figs 3 and 4 show a typical representation of the endarterectomized surface with and without topical saratin with scanning electron microscopy at 2000× magnification.
Fig. 3.
Electron micrograph (2000×) of endarterectomized rat carotid artery 3 hours after carotid endarterectomy. A, Control surface. B, Surface receiving topical saratin (5 μL). Control surface shows abundant cellular elements including fibrin strands, red blood cells, and platelets. Saratin-treated surface shows marked decrease in cellular elements.
Fig. 4.
Electron micrograph (2000×) of endarterectomized rat carotid artery 24 hours after carotid endarterectomy. A, Control surface. B, Surface receiving topical saratin (5 μL). Control surface shows numerous red blood cells and platelets. Saratin- treated surface shows marked decrease in platelet adhesion.
The application of topical saratin after a carotid endarterectomy significantly decreased the development of intimal hyperplasia as compared with the control group. In terms of luminal stenosis, control rats showed a 29.8% luminal stenosis versus a 10.9% luminal stenosis in the saratin-treated group (Fig 5).
Fig. 5.
Percent luminal stenosis resulting from intimal hyperplasia 2 weeks after carotid endarterectomy. Saratin-treated group (n = 15) and control group (n = 10) are shown. Saratin group received 5 μL of saratin applied topically to exposed subendothelial surface. Star indicates P value of .004.
For platelet counts to be determined, it was necessary to obtain a preoperative blood sample from each rat of approximately 1- to 1.5-cc, which represents a significant portion of a rat's total blood volume. In view of this, it is reasonable to expect a decrease in the postoperative platelet counts of both control and saratin rats. The difference between preoperative and postoperative platelet counts was compared for the control and saratin-treated groups. No statistical significance in the platelet count differences between control and saratin-treated rats was noted. No statistically significant differences were observed between preoperative and postoperative bleeding times.
Discussion
Carotid endarterectomy is one of the most common vascular surgical procedures performed in the United States. Results from multicenter trials have demonstrated the efficacy of this procedure in the treatment of extracranial carotid disease in patients both with and without symptoms.5, 6 Endarterectomy procedures are used in the treatment of occlusive vascular disease in other vascular beds.7 Endarterectomy leaves a significant area of thrombogenic subendothelium exposed. Despite the efficacy of carotid endarterectomy, the operation can lead to complications, including thrombosis and the development of intimal hyperplasia. Platelet accumulation may occur at the endarterectomy site. Clinical studies have demonstrated a post–carotid endarterectomy stroke rate of 1% to 10% in the immediate postoperative period, most of which is accounted for by thrombus formation with subsequent cerebral embolization.1 Platelet accumulation may also result in restenosis due to intimal hyperplasia. Restenosis has been reported to occur in 10% to 20% of endarterectomized patients at 2 to 5 years postoperatively, most of which is due to intimal hyperplasia, intimal thickening, and vessel diameter reduction.2 In the current study we have demonstrated a significant reduction in platelet adhesion after carotid endarterectomy that is associated with the clinically relevant finding of reduced intimal hyperplasia. Saratin-treated rats showed only 25% of the intimal hyperplasia observed in control rats.
Intimal hyperplasia development involves medial smooth muscle cell proliferation, migration into the intima, and finally proliferation and production of extracellular matrix.8 Studies in which endarterectomized rat carotid artery models9 and embolectomy balloon-injury models10 have been used have shown that as the endothelial cells are stripped away during vascular injury, platelets begin to adhere to the exposed subendothelium. Spallone et al,9 using scanning electron microscopy, have shown that 5 minutes after a carotid endarterectomy in a rat, a monolayer of platelets is formed over the injured area. Fifteen minutes after the injury, platelet aggregation and thrombus formation are observed. Thirty minutes after endarterectomy, the site is covered with activated platelets and coated by fibrin and red blood cells. Thrombus formation reaches its peak at 3 hours after injury, with a thick fibrin-platelet layer being observed. Platelets are an integral component of this thrombus formation and thus thrombosis, but they also appear to play a role in the development of intimal hyperplasia. Studies in which thrombocytopenic rats have been used have demonstrated a significant decrease in intimal thickening after carotid artery injury as compared with control rats.11 Once platelets adhere to the exposed subendothelium of an injured vessel, they become activated and release their granules. These granules contain vasoactive and thrombotic factors (serotonin, adenosine diphosphate, fibrinogen, von Willebrand factor, thromboxane A2), as well as growth factors (platelet-derived growth factor, transforming growth factor-β, and epidermal growth factor).12 The exact mechanisms by which platelets enhance the development of intimal hyperplasia are not yet completely understood. Studies suggest that platelets provide primarily a chemotactic stimulus for medial smooth muscle cell migration toward the intima during the second phase of intimal hyperplasia development.13 Other studies, in which anti–platelet-derived growth factor antibodies were used, have demonstrated the vital role that platelet-derived growth factor plays in neointimal smooth muscle cell accumulation after a vascular injury.14 Another mechanism by which platelets may enhance the development of intimal hyperplasia is through the activation of the coagulation cascade and the subsequent accumulation of thrombin at the site of injury. Several studies have demonstrated the mitogenic effects of thrombin on smooth muscle cells.15, 16 In addition, thrombin has been shown to be a stimulus for platelet activation.17 Regardless of the precise mechanism, platelet adhesion and activation at the site of a vascular injury play a significant role in the development of thrombosis and intimal hyperplasia, and therefore, inhibition of platelet adhesion and activation may help prevent or reduce thrombosis rates and intimal hyperplasia development. An ideal agent for the attenuation of the intimal hyperplasia response would be one that produces sitespecific and localized antiplatelet effects without systemic distribution or a generalized coagulopathy.
It appears that the vital steps that precipitate the cascade of events leading to thrombosis and later intimal hyperplasia stem from the interaction between the exposed subendothelial collagen at the site of vessel injury and a monolayer of platelets that adhere to the exposed collagen. A specific inhibitor of this platelet to subendothelial collagen adhesion may serve to prevent or at least decrease the development of thrombus and intimal hyperplasia. This study was designed to examine one such agent. The novel aspect of the study is the use of a topical agent at the site of injury with the potential of decreasing thrombosis, intimal hyperplasia, or both without any systemic effects. A rat carotid endarterectomy model was used that is reliable and reproducible and that mimics a human carotid endarterectomy to a closer extent than the balloon-injury model.3 We think that the use of a direct arteriotomy and suture closure is crucial for evaluation of an agent that may lead to a reduction in localized hemostatic mechanisms.
Studies with hematophagous organisms have shown that salivary secretions from various leeches have antiplatelet properties. Leech antiplatelet protein, a protein of approximately 16,000-d from the leech Haementeria officinalis has been shown to inhibit collagen-induced platelet aggregation and adhesion, as has Calin, a 65,000-d protein from H medicinalis, 18, 19 and leech antiplatelet protein and Calin have been shown to inhibit von Willebrand factor binding to collagen.20, 21 However, in the most pathophysiologically relevant situations (mechanical damage to the arterial wall, plaque rupture), platelet adhesion through collagen–von Willebrand factor interaction is thought to be crucial in the initial tethering of platelets (through platelet glycoprotein Ib/IX/V receptors). Therefore, focused inhibition of platelet adhesion through collagen–von Willebrand factor binding, with little or no effect on platelet aggregation, might result in a more localized, injury-specific antithrombotic response, without general systemic antiplatelet effects. Thus, the saliva of H medicinalis was analyzed for the existence of a selective inhibitor of collagen–von Willebrand factor binding. Indeed, a protein of relatively low molecular weight (approximately 12,000 d), whose primary mechanism of action is thought to be inhibition of von Willebrand factor binding to collagen at high shear and which has no effect on platelet aggregation at these concentrations, has been isolated and named saratin . This saratin-mediated inhibition of initial platelet adhesion under elevated shear may result in a decreased thrombosis rate and a decrease in the development of intimal hyperplasia. This represents a modality with specific and localized effects, well suited for application by both surgeons and interventional radiologists.
We have shown a significant decrease in platelet adhesion and accumulation after a vascular injury similar to endarterectomy. The decreased platelet adhesion is seen both immediately after endarterectomy (3 hours) as well as at 24 hours. The effect at 24 hours is significant in that we think this effect is not due to the direct inhibitory effect of saratin on the collagen; rather, it is due to the initial inhibition of platelet aggregation and the subsequent disruption of the platelet activation cascade. Once platelets are initially inhibited from attaching to exposed collagen, the platelet cascade cannot proceed. Fig. 1, Fig. 2 demonstrate that the topical application of saratin on the recently injured vessel can inhibit platelet adhesion to a significant degree. Saratin inhibited platelet adhesion by 59% and 77% at 3 and 24 hours, respectively. This inhibition is noted in the visible differences in cellular element deposition between control and saratin-treated arteries at both 3 hours and 24 hours (Fig. 4, Fig. 5). We propose that this lack of cellular response is due to the inhibition of platelet adhesion. This represents a unique mode of therapy for the inhibition of platelet adhesion to an injured vessel. Control rats had a significantly reduced luminal diameter at 2 weeks after carotid endarterectomy compared with saratin-treated rats. The amount of intimal hyperplasia development was significantly reduced in saratin-treated rats, which correlated with reduced platelet adhesion and accumulation. The finding of decreased intimal hyperplasia and thrombosis provides for clinically relevant end points and sequelae of reduced platelet adhesion. This suggests that the site-specific, nonsystemic inhibition of platelet aggregation and adhesion can lead to a decreased thrombosis and occlusion rate and possibly reduce the incidence of cerebrovascular events that occurred after carotid endarterectomy. The degree or level of intimal hyperplasia and luminal stenosis improvement may be arguable; however, the significant finding of a decreased thrombosis rate in rats treated with saratin is relevant. A full 33% of control rats showed thrombosis, whereas none of the saratin-treated carotid arteries were thrombosed. Of particular clinical importance is the lack of systemic effects demonstrated by this agent. The local application of saratin did not affect systemic bleeding times or platelet counts when compared with controls. This implies that the decreased platelet adhesion and the subsequent decrease in thrombosis and intimal hyperplasia are the result of local effects. These findings are encouraging and elicit a cautious degree of excitement in that the spectrum of clinical applications for a modality that is capable of locally inhibiting the deleterious effects of platelet adhesion and activation without disturbing the systemic hemostatic mechanism is vast.
Acknowledgements
We wish to thank Kim Henning, BS, LATG and Mr Wasson S. Snow, LATG, Supervisor of the Veterinary Medical Unit at the Central Arkansas Veterans Health Care System and Cindy L. Hastings, BS, Lead Technologist of electron microscopy for their invaluable assistance in this study. We would like to thank Merck KGaA for funding this study and for supplying the saratin required for our investigations. Finally, we would like to thank Ethicon, Inc, for supplying the suture required for the surgical procedures.
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☆ Competition of interest: nil.
☆☆ Funded in part by Merck KGaA, Hornick Endowment Fund University of Arkansas for Medical Sciences.
★ Reprint requests: Mohammed M. Moursi, MD, Department of Surgery, Division of Vascular Surgery, University of Arkansas for Medical Sciences, Slot 520, 4301 West Markham St, Little Rock, AR 72205 (e-mail: MoursiMohammedM@exchange.uams.edu ).
PII: S0741-5214(01)24605-9
doi:10.1067/mva.2001.116801
© 2001 Society for Vascular Surgery and The American Association for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
