ABSTRACT
Purpose: The perioperative management of patients on antithrombotic therapy is currently an unresolved problem as these therapies pose a considerable risk for perioperative hemorrhagic complications. The presented studies investigated the efficacy of a new collagen technology to achieve hemostasis. A polyethylene glycol-coated collagen pad (PCC) was compared to a marketed fibrinogen-thrombin coated collagen pad (FTC) for the treatment of an aortotomy incision in heparinized swine on dual antiplatelet therapy. Materials and Methods: Twenty-eight 3-mm aortotomy incisions were created in nine heparinized pigs without antiplatelet therapy and treated with PCC. Sixty-eight aortotomy incisions were created in ten heparinized pigs that received clopidogrel (10–11 mg/kg) and acetylsalicylic acid (8–11 mg/kg) orally for 5 days, and treated with either PCC or FTC (N = 34/group). Dual antiplatelet therapy resulted in significantly reduced platelet function. Aortotomy incisions resulted in life-threatening bleeding of 35–292 ml/min. Results: In animals without antiplatelet treatment, PCC provided 96% immediate hemostatic success. In animals with antiplatelet treatment, FTC provided 18% immediate hemostatic success increasing to 74% after 10 min. Strikingly, PCC provided 94% immediate success increasing to 100% after 10 min. Controlling for differences in pretreatment bleeding rates, statistical model-estimated time to hemostasis was 12 times shorter in PCC-treated lesions (p < .02). Conclusion: The combination of a procoagulant collagen pad with a synthetic sealing component provides excellent hemostatic properties under a worst-case scenario. PCC rapidly and firmly adheres to tissue, thereby controlling severe arterial bleeding, even when platelet function is significantly reduced. Treatment with PCC provided superior time to hemostasis compared to FTC.
INTRODUCTION
The use of antiplatelet agents for the prevention of cerebrovascular and cardiovascular disease is a well-established practice and has reduced the incidence of cardiovascular events, including myocardial infarction and stroke [Citation1]. Treatment with the P2Y12 receptor antagonist clopidogrel and acetylsalicylic acid (ASA) can be considered a standard therapy for ischemic heart disease [Citation2]. By reducing or depleting platelet function, antiplatelet therapy prevents thrombotic events but poses a significant risk for major bleeding complications in planned cardiac interventions as well as in elective and emergency non-cardiac surgeries [Citation3–5]. Topical hemostats and sealants can be used to mitigate such bleeding risks [Citation6].
A new topical hemostat, a polyethylene-glycol coated collagen pad (PCC), has recently been described [Citation7]. PCC is a soft, thin, pliable bovine collagen pad coated with a protein-reactive polyethylene-glycol (PEG) cross-linker that promotes hemostasis and strong tissue adherence. Collagen induces clot formation through platelet activation which could be negatively influenced by antiplatelet therapy. However, PEG covalently binds the pad to proteins in blood and on the tissue surface and seals the wound.
Based on this dual mechanism of PCC, we hypothesize that antiplatelet therapy does not influence the efficacy of PCC. There are no previous preclinical or clinical reports systematically evaluating a ready to use, synthetic-collagen dual mechanism patch for control of high pressure/high volume hemorrhages without applying any other methods to achieve hemostasis. Further, to our knowledge, no preclinical model investigating the efficacy of hemostatic agents in a dual-antiplatelet therapy model has been published. Therefore, a prospective randomized study was conducted to assess the efficacy of PCC compared with an equine collagen patch coated with human coagulation factors (FTC) in a clinically relevant porcine severe aortic bleeding model treated with dual antiplatelet therapy.
MATERIALS AND METHODS
Hemostatic Agents
Hemopatch [Sealing Hemostat] (Baxter AG, Vienna, Austria) is an absorbable pentaerythritol polyethylene glycol ether tetra-succinimidyl glutarate (NHS-PEG) coated bovine collagen pad (PCC). The nonactive side of the pad is marked with blue squares for differentiation. TachoSil [Absorbable Fibrin Sealant Patch] (Takeda Austria GmbH, Linz, Austria) is a human fibrinogen (5.5 mg/cm2) and human thrombin (2.0 IU/cm2) coated equine collagen pad (FTC). The active side of the pad is marked with yellow for differentiation. Both products were stored and handled according to their instructions for use or product insert [Citation8, 9].
Scanning Electron Microscopy (SEM)
Dry, naïve pieces of each hemostatic agent were characterized using scanning electron microscopy (SEM). After being removed from their packaging, each hemostat was cut into smaller specimens with a razor blade, mounted onto aluminum supports, coated with metal to enhance conductance and examined in the SEM (JSM 7600F Thermal Field Emission SEM, JEOL, Peabody, MA).
Experimental Animals
Animal procedures were approved by the local Institutional Animal Care and Use Committee and conducted in accordance with the Guide for the Care and Use of Laboratory Animals and applicable United States animal welfare regulations in an AAALAC-accredited facility [Citation10].
The two studies were conducted using a total of 19 male, domestic pigs, weighing 56–67 kg at the time of surgery. Animals were quarantined for at least 6 days upon arrival and only animals showing no signs of clinical illness were used. Animals were socially housed in stainless-steel pens in a humidity and temperature-monitored room. The rooms were maintained at 18–21°C, 30–70% humidity, and a 12-hr light/dark cycle. Pigs received water ad libitum and a standard pig diet once daily.
Experimental Groups
Two experiments were conducted: the first study used 9 animals that did not receive antiplatelet therapy and the second study used 10 animals that did receive antiplatelet therapy. In the first study, hemostasis of PCC (N = 28) was evaluated. In the second study, hemostasis of PCC and FTC (N = 34 per group) was compared.
Preparation and Monitoring
Anesthesia for all animals was induced with up to 5% isoflurane delivered in a 2:1 nitrous oxide:oxygen carrier to facilitate intubation. Ophthalmic ointment was applied to both eyes and the left thorax was prepared for aseptic surgery. Anesthesia and analgesia during hemostasis evaluation was maintained with ketamine (25–30 mg/kg/hr, IV) and fentanyl (20 mcg/kg/hr, IV), and up to 1% isoflurane. Lidocaine (2 mg/kg, IV) was administered after thoracotomy to prevent cardiac arrhythmias. Analgesia provided by ketamine and fentanyl during the surgical procedures was deemed sufficient after consultation with the staff veterinarian. Therefore, no additional analgesics were administered. Antibiotics were found not to be indicated as aseptic techniques were applied for this acute terminal procedure. Pigs were mechanically ventilated with 100% oxygen and respiratory minute volume was adjusted to maintain the highest end-tidal CO2 at which the animals did not breathe spontaneously. Average peripheral oxygen saturation was 98%. A jugular vein catheter was placed for intraoperative blood sampling and a carotid artery catheter for monitoring blood pressure and heart rate. Pigs received a preemptive dose of PlasmaLyte A (300 ml, IV bolus) followed by a continuous infusion (10 ml/kg/hr, IV). Intravenous fluid therapy was adjusted based on blood pressure trends. Additionally, dobutamine (up to 2 mcg/kg/min, IV) was given to maintain mean arterial blood pressure above 75 mmHg and atropine (0.02 mg/kg, IV) was given to counteract a decrease in heart rate as needed. Plasma total protein, as estimated by refractometry, and hematocrit were measured approximately every 20 min to monitor for hemodilution.
A baseline activated clotting time (ACT) was measured using a HEMOCHRON Jr. Signature+ Whole Blood Microcoagulation System (ITC, Edison, NJ) prior to the surgical procedure followed by heparin administration (initial 90 U/kg IV bolus followed by up to 50 U/kg/h). ACT was monitored throughout the surgical procedure prior to each hemostasis evaluation and additional heparin was administered as needed to achieve an ACT of approximately 2X baseline (161–219 s).
Surgical Model
The surgical model was performed as previously described by Agger et al. [Citation11]. Briefly, a left thoracotomy was performed at the sixth intracostal space to gain access to the entire length of the descending thoracic aorta. A series of up to seven, 3-mm long, longitudinally oriented lesions were created starting distal to the aortic arch using a custom-made lesion sizing device. Lesion size was confirmed by measuring the exposed blade width with calipers. A quantitative assessment of bleeding was performed for each lesion pretreatment by using preweighed gauze to absorb blood from the lesion for 6 s after which it was weighed again. The difference in pre- and posttreatment weights was multiplied by 10 to calculate a bleeding rate in ml/min, assuming 1 g of blood equals 1 ml. Lesions were only created if the systolic blood pressure was between 95 and 105 mmHg to reduce potential bias in bleeding severity secondary to differences in blood pressure. PCC and FTC were cut into 3 × 2.5 cm squares, so that they overlapped the margin of the lesion by approximately 1 cm. Treatment of individual lesions with PCC or FTC was randomized and the surgeon was blinded to treatment until after the lesion was created. PCC and FTC were applied dry with the appropriate side in contact with the lesion and surrounding aortic tissue. At 2 min (PCC) or at 3 min (FTC) after application, per instruction for use for each product, pressure was released leaving the hemostat in contact with the aorta. Following the last hemostasis assessment, animals were humanely euthanized while under deep ketamine/fentanyl anesthesia by exsanguination.
Hemostasis Evaluation
Hemostasis was evaluated qualitatively immediately after the approximation time and then every 30 s until 10 min after application. Hemostatic effectiveness was defined as flush adherence to the aorta and no observed bleeding, either through or from the margins of the pad. If hemostasis was not achieved, pressure using gauze held in ring forceps was reapplied to the pad until the next evaluation time point. If hemostasis was not achieved within 10 min after application, the lesion was treated by alternative means (e.g., suture, application of additional hemostats). Hematomas that developed under the pads (i.e., delamination of the pad from the tissue surface) were assessed as hemostatic failure and pressure was reapplied as described above and consistent with Agger et al. (Personal communication, September 23, 2013).
Dual Antiplatelet Therapy
Animals in the antiplatelet therapy groups received a daily oral dose of 8–11 mg/kg clopidogrel and 10–11 mg/kg acetylsalicylic acid (ASA) starting 4 days before and on the day of hemostasis evaluation. The last oral dose (day 5) was given at least 1 hr before induction of anesthesia. Blood samples were collected in 3.2% trisodium citrate solution prior to antiplatelet therapy (day 0) and again after the last administration of drugs (day 5). To confirm adequacy of antiplatelet therapy, platelet aggregation in platelet-rich plasma (PRP) triggered by collagen (6 μg/ml) and adenosine diphosphate (ADP, 10 μmol/l) was measured by turbidometric method (Model 700 Lumi-Aggregometer, Chrono-Log) as previously reported [Citation12]. The platelet count was determined in the PRP sample and adjusted to 250×103/μl with homologous platelet-poor plasma. Aggregation was expressed as the maximal percentage change in light transmittance from baseline with platelet-poor plasma as a reference based on duplicate measurements of each sample.
Histopathology
Aorta sections were collected immediately following euthanasia, fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. Slides were evaluated under light microscopy at 10× and imaged using an Aperio ScanScope CS (Leica Biosystems Inc., Buffalo Grove, Illinois, USA).
Statistical Analysis
All calculations were performed with the software R version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria) [Citation13]. The level of statistical significance was set to 5%.
Inhibition of collagen- and ADP-induced platelet aggregation in PRP was compared between pre- (day 0) and posttreatment (day 5) in animals receiving antiplatelet therapy using a t-test for paired samples. Interval censored time to hemostasis was the primary endpoint. The distributions of time to hemostasis were displayed by animal and hemostatic agent using Kaplan–Meier plots [Citation14]. An accelerated failure time model was used to model the difference in interval-censored time to hemostasis between PCC and FTC. The model consisted of fixed effects covariates: (1) hemostatic agent (PCC or FTC) and (2) pretreatment bleeding rate in ml/min. The model was fitted using R function “survreg” of R package survival [Citation15]. Random effects for factor “animal” and “agent” crossed with “animal” were included by R function “frailty” of the same R package. Differences in time to hemostasis between PCC and FTC were assessed by their ratio, a corresponding two-sided 95% confidence interval (CI) and a two-sided p-value for the alternative hypothesis of a difference in time to hemostasis between PCC and FTC. A comparison of the primary endpoint between PCC-treated animals with normal platelet function and animals with severely decreased platelet function based on Kaplan–Meier estimates, was carried out using a log-rank test for two independent samples. Fischer's exact test was used to compare hemostasis at 3 and 10 min after application, and for hematoma formation under each pad in antiplatelet treated animals.
Figure 1 Scanning electron microscopy (SEM) photos of a polyethylene glycol coated collagen pad (PCC, Hemopatch) and a fibrin and thrombin coated collagen pad (FTC, TachoSil). (A, B) Cross sectional images show the open porous structure of PCC compared to (C, D) the closed-cell honeycomb like matrix of FTC. (A) The NHS-PEG coating on PCC and (C) the human protein coating on FTC are on the right surface (arrows). (A, C) Lower magnification (B, D) higher magnification.
RESULTS
A SEM comparison of cross-sections of the hemostatic pads, PCC and FTC, as seen when removed from their packaging (dry) is shown in Figures –D. The images depict the clear structural differences of the collagen matrix of the two products. While PCC (Figures and B) has an open porous structure, the FTC (Figures and D) collagen pad appears to have a closed honeycomb-like structure.
Ex vivo collagen- (Figure ) and ADP-induced platelet (Figure ) aggregation in PRP was significantly inhibited (p < .001) by the daily oral treatment with clopidogrel and ASA. Mean aggregation response to collagen and ADP after dual antiplatelet therapy for 5 days was reduced by 45% and 31%, respectively.
Figure 2 Clopidogrel and acetylsalicylic acid (ASA) statistically significantly inhibited platelet function (p < .001). Platelet aggregation in PRP was assessed using (A) a collagen- and (B) adenosine diphosphate (ADP)-triggered platelet aggregation assay. Results expressed as the mean of duplicate measurements for individual animals before (white bars) and after (black bars) a 5-day administration of antiplatelet therapy are shown.
For PCC-treated lesions, the median pretreatment bleeding rate was 164 (Range: 55–292, N = 28) ml/min in animals not on antiplatelet therapy and 130 (54–251, N = 34) ml/min in animals on antiplatelet therapy. The overall median pretreatment bleeding rate for PCC-treated animals was 150 ml/min (N = 62). For FTC-treated lesions in animals receiving antiplatelet therapy, the median pretreatment bleeding rate was 117 (35–237, N = 34). The overall median pretreatment bleeding rate for all lesions in animals on antiplatelet therapy was 127 ml/min (N = 68).
For PCC-treated lesions, hemostatic success within the 10-min observation period was 100% whether animals were on platelet therapy or not (N = 62 lesions; Figures and B); whereas, for FTC-treated lesions, hemostatic success was evident in 25/34 (74%) lesions within the 10-min observation period (Figure ). PCC provided 96% hemostatic success without antiplatelet therapy and 94% success with antiplatelet therapy within 2 min of application, the minimum manufacturer recommended application time for PCC. The success rate of PCC was increased to 100% (no antiplatelet therapy) and 97% (antiplatelet therapy) within 3 min, the minimum manufacturer recommended application time for FTC. In contrast, FTC provided 18% hemostatic success within 3 min (Table ).
Figure 3 Superior hemostatic properties of PCC, a polyethylene glycol coated collagen pad relative to FTC, a fibrinogen-thrombin coated collagen pad. (A) Kaplan-Meier plots for interval-censored time to hemostasis in animals treated with PCC without antiplatelet therapy; (B) Kaplan–Meier plots for interval-censored time to hemostasis (PCC, solid line; FTC, dashed line) per animal on dual antiplatelet therapy; grey boxes represent time interval during which hemostasis was achieved (3–4 lesions per group per animal).
TABLE 1 Hemostasis properties of PCC versus FTC
Delamination of the pad from the tissue surface was evident by hematoma formation in 1/28 (4%) lesions for PCC in animals receiving no antiplatelet therapy and was not observed in animals on the antiplatelet therapy (0/34; 0%). Hematoma formation was observed in 26/34 (76%) lesions for FTC (Table ).
The time to hemostasis was not statistically significantly different for lesions treated with PCC between animals with normal platelet function and animals with severely decreased platelet function (Figure , p = .4648). However, compared to lesions treated with FTC the time to hemostasis was 12 times shorter with PCC in animals with severely decreased platelet function (Figure , p = .01987; 95% CI: 1–102). The calculated probability for achieving hemostasis over time for a median pretreatment bleeding rate of 127 ml/min illustrates the large difference in hemostatic efficacy between PCC and FTC (Figure ).
Figure 4 (A) Statistical model-estimated probability of hemostasis over time with PCC, a polyethylene glycol coated collagen pad, with (solid line) and without (dashed line) antiplatelet therapy calculated based on a median bleeding rate of 150 ml. (B) Statistical model-estimated probability of hemostasis over time with PCC (solid line) relative to FTC, a fibrinogen-thrombin coated collagen pad (dashed line) calculated based on the median bleeding rate of 127 ml/min in animals on dual antiplatelet therapy. Time to hemostasis was 12 times shorter (95% CI: 1 to 102) with PCC than with FTC.
Collagen pad application during surgery is depicted in Figure . Histological sections of a representative aorta at the level of incisions after treatment illustrate the strong and tight tissue adherence of PCC (Figure ) relative to FTC. An increased sub- and intrapatch blood accumulation can be observed with FTC (Figure ).
Figure 5 Sutureless aortotomy repair. (A) Hemostatic agents 10 min after application in situ during surgery (left is cranial and top is dorsal; cranial pad is FTC and caudal pad is PCC); Hematoma formation over incision (arrows). (B, C) Representative images of hematoxylin and eosin-stained aortic cross sections; x10. (B) Clot formation (asterisk), flush adherence to aortic tissue, and tight sealing with PCC. (C) Clot formation (asterisk), and increased sub- and intrapatch blood accumulation (arrows) with FTC.
DISCUSSION
The present report analyzed the impact of dual antiplatelet therapy on the efficacy of a sealant hemostat in a severe arterial bleeding model. Based on our results, significant reduction in platelet function, using clopidogrel and ASA for dual antiplatelet therapy, did not affect the performance of PCC. Time to hemostasis was not different between animals with and without antiplatelet therapy. Hemostasis was reached in 97% of lesions within 3 min after application with PCC. The rapid time to hemostasis was in stark contrast to the performance of FTC, where only 18% of lesions were successfully treated within 3 min.
The pig is generally accepted as a model to evaluate hemostatic efficacy due to its similar anatomy and physiology to humans [Citation16]. In an overview of animal models for vascular surgery, Byrom et al. concluded that porcine arterial morphology, platelet function, and coagulation all show similarities to humans [Citation17]. The clinical relevance of the pig as a vascular model in this study was further refined by using clinically effective clopidogrel and ASA dosages, which were confirmed by a reduction of platelet function ex vivo using collagen- and ADP-induced platelet aggregation in PRP. While dual antiplatelet therapy inhibits clot formation, it did not affect the severity of the arterial injury. Bleeding rates were above 35 ml/min in all lesions, which is well above what can be considered life-threatening hemorrhage [Citation18]. A lower pretreatment bleeding rate in animals receiving clopidogrel and ASA was observed; however, this is likely due to normal variation in bleeding and minor systematic imprecision in the collection of spurting blood. FTC was selected as a comparator, as it has been investigated for use in sutureless coronary artery [Citation19] and ventricular wall repair [Citation20] and in a similar porcine aortotomy model [Citation11].
PCC was previously evaluated in several clinically relevant preclinical hepatic and vascular bleeding models with mild to moderate bleeding showing superior performance when compared to FTC and oxidized cellulose [Citation21, 22]. Consistent with a preclinical report investigating FTC in a heparinized swine spleen incision model, a high adhesive failure rate was observed in the present study as was evident in the histology images and by hematoma formation in 76% of lesions treated with FTC compared to no observed hematoma in the PCC antiplatelet group. Matonick and Hammond [Citation23] attributed the high incidence of adhesive failure of FTC in the spleen incision model to the structural design of the pad. The closed cell honeycomb-like structure of FTC inhibits blood flow within the matrix, resulting in increasing blood applied stress levels occurring at the matrix-to-tissue interface. As suggested by Matonick and Hammond, a three-dimensional porous structure that allows the migration of blood through the material, therefore, reducing the blood-tissue interface stress level is responsible for improved adherence of hemostats. Cross-sectional imaging demonstrates the difference between PCC and FTC, with PCC having an advantageous open porous collagen structure yielding excellent absorption capacity, thereby reducing the likelihood of being washed away.
In situations with normally functioning platelets, the collagen component of PCC rapidly induces coagulation through direct binding of platelets via the platelet receptor GPVI and integrin α2β1, and indirect binding of platelets through binding of von Willebrand factor which, in turn, binds the platelet receptor GPIb-IX-V and integrin αIIbβIII [Citation24, 25]. Receptor and integrin binding results in intracellular signaling events that reinforce platelet adhesion and mediate platelet activation, leading to platelet aggregation and a growing clot. The platelet inhibitors, clopidogrel and ASA, inhibit platelet activation by interfering with specific signaling pathways, and, therefore, potentially reduce the collagen mediated hemostatic effects of PCC. However, the collagen matrix of PCC functions as the vehicle to deliver NHS-PEG to the bleeding surface.
When platelet function is severely reduced, rapid coagulation cascade-independent sealing becomes crucial. NHS-PEG is hydrolyzed upon contact with blood and other body fluids. The resulting molecular components covalently bind with blood proteins and the tissue surface to which the pad is applied. The NHS esters of NHS-PEG readily bind the ϵ-amino group of lysine residues and the N-terminal α-amino group of proteins [Citation26]. PEG hydrogel sealants have previously been observed to have good adherence to biological tissue and to be effective in vivo [Citation27].
While our present results suggest greater efficacy with PCC compared to FTC in high pressure and high volume bleeding, human studies are necessary to verify these results. To date, there are no data from large clinical studies, but several case reports describe the use of PCC to treat bleeding in patients [Citation28–31]. Interestingly, Erdas et al. demonstrated in a randomized controlled pilot trial that FTC is not superior to standard hemostasis (ligation and use of a harmonic scalpel) in preventing postoperative bleeding risk in patients on antithrombotic therapy with vitamin K antagonists or ASA undergoing thyroid surgery [Citation32]. These findings highlight a limitation of this study, which is a lack of long term follow up. Aoyagi et al. reported successful intraoperative sutureless repair for an oozing type bleeding of a free wall ventricular rupture using FTC but recurrent ventricular wall rupture and a ventricular aneurysm were observed postoperatively in two out of three patients [Citation33].
The present study was the first investigation comparing PCC and FTC in treating severe arterial bleeding using a heparinized porcine aortotomy model with significantly reduced platelet function. PCC provided statistically superior hemostasis relative to FTC that was attributed to its strong and rapid adherence to the underlying tissue, as was evident by a lower incidence of hematomas. The results are consistent with other preclinical studies and clinical reports investigating the efficacy of PCC. Though clinical studies are needed, this study demonstrates that PCC may be an effective hemostatic agent in patients being treated with antiplatelet therapies throughout the peri- and intraoperative period.
Financial disclosure
The study was supported by Baxter Healthcare Corporation, 1 Baxter Parkway, Deerfield, IL 60015. The authors are employees of Baxter Healthcare Corporation.
ACKNOWLEDGMENTS
The authors thank Dr. Tracy Carlson for histopathological support, Mary Ann Murphy and Jim Diorio for SEM images, Drs. Jeff McKee, Gary Leung, and Joe Safron for the surgical model development support, and Dr Heinz Gulle for critical review of the manuscript. The authors thank their technical and administrative staffs.
Declaration of interest: The authors report conflicts of interest. Studies were designed and performed using sound scientific methods and standardized lesions for impartial data collection and comparison. The authors alone are responsible for the content and writing of the article.
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