Wound Pharmacobiology

ABSTRACT

Paul C. Peters Jr, MD

Wound healing after major joint surgery involves a series of complex events. Over the past several years, thrombin has emerged as a pivotal participant in wound healing. The high incidence of venous thromboembolism following major joint replacement has made prophylaxis with pharmacologic agents a component of postoperative management. Anticoagulant agents of various classes affect different degrees of thrombin inhibition by virtue of their in vivo mechanisms of action. By inhibiting thrombin activity directly, with or without antithrombin III or other antithrombins, traditional anticoagulants may retard the wound healing process and impair completion. Newer, more selective anticoagulants may provide not only more effective alternatives for venous thromboembolism prophylaxis surrounding major orthopedic procedures, but also an anticoagulant environment more favorable to wound healing.

Wound healing is a complex cascade of events requiring carefully scripted interactions among various cell types and the participation of extracellular matrix molecules and numerous cellular growth factors. Platelets, macrophages, fibroblasts, epidermal cells, and endothelial cells play a significant role in wound healing. Each has mechanisms that include cellular activation with up-regulation of surface receptors for growth factors (eg, ?-fibroblast growth factor, transforming growth factor- ?, and platelet-derived growth factor) or for coagulation factor VII and others such as tissue factor. Specific adhesion molecules on the surfaces of activated platelets, white cells, and macrophages localize repair processes. Repair processes are fostered by the secretion of cellular “glue” proteins such as von Willebrand factor and fibrinogen (the latter is also the central structural protein of clotted blood), as well as growth factors that promote activation, migration, and proliferation of fibroblasts, endothelial cells, and epithelial cells.

Wound healing is most efficient when injury to tissue and blood vessels occurs in a fully functional clotting system in which platelets are plentiful and well endowed, glue proteins are adequate, fibrin cross-linking to other fibrin molecules as well as to fibronectin and other matrix components is rapid and unhindered, and activation of the fibrinolytic system is suitably delayed in onset.

Grades of Wound Repair

Three grades of wound repair have been described in the literature. The first grade, healing by primary intent, is seen in simple small incisions, usually closed mechanically, in which inflammation plays a minimal part and wound healing is rapid.

The second grade, healing by secondary intent, occurs in deep or open wounds and involves tissue loss and inflammation. The wound, left unsutured, undergoes contraction of its margins, increasing proximity of these margins and diminishing the area where repair is required. Re-epithelialization occurs and scar tissue eventually forms, undergoing remodeling over time.

The third grade, healing by tertiary intent, is seen in open, infected wounds that do not show the same steps observed in healing by secondary intent. Several interactive mechanisms are involved in wound healing by secondary intent. Inflammation, granulation tissue formation, and collagen remodeling are the most prominent processes and, because of the interfaces between them, the three phases often overlap. As suggested above, these phases/grades of wound repair are modulated by an extensive number of biochemical mediators. The extent and severity of the wound, host hemostatic and immunologic integrity, and environmental conditions at the wound site determine the time needed for healing of the wound, as well as the quality of the repair.^1-3

The initial response to an acute wound is activation of hemostasis. Blood exposure to the injured endothelium results in platelet adhesion and activation and the initiation of the coagulation cascade. The coagulation cascade culminates in the formation of a fibrin matrix that provides a “lattice” for the transport of various cells into the wound.¹ Over the past several years, thrombin, the culmination of the cascade’s effort, and its master enzyme has emerged as a critical contributor to wound healing.^4-6

Although the need for protection against venous thromboembolism is greater in major orthopedic surgery than in other clinical situations, the process of wound repair may be seriously impaired by anticoagulants administered for thrombosis prophylaxis. Timely wound healing and avoidance of complications, such as wound bleeding, prolonged or excessive wound drainage, and infection of the wound, or — by extension — infections of the prosthetic device, are major considerations in patients undergoing orthopedic surgery. This paper discusses the role of thrombin and the potential impact on wound healing of antithrombotic agents used for prevention of venous thrombosis.

The Role of Thrombin in Wound Healing

The first step in wound healing entails the formation of a hemostatic plug with thrombin playing a central role in the process. Thrombin exhibits several other functions in wound healing: it behaves as a growth factor, stimulating mitogenesis in fibroblasts and epithelial cells and triggering or enhancing growth factor production in various cell types; up-regulates collagen production and promotes contraction of aggregated cells as an element of wound closure; and improves vascularization of healing tissue.^5,7-16 In animal models, thrombin or peptide portions of thrombin significantly increase the rate of wound healing of full thickness incisions.^10,16-17

Many of thrombin’s effects on wound healing are mediated through proteinase-activated receptors.^7-10 In some patients, thrombin binding to proteinase-activated receptors on the surface of fibroblasts, epithelial cells, keratinocytes, or monocytes is sufficient for effective wound closure. In other instances, the proteolytic activity of thrombin also is required for stimulation. In situations where binding activity alone is sufficient, peptide fragments of thrombin effectively mimic the actions of the full molecule.

Various animal studies have demonstrated that physiological levels of thrombin peptide fragments can significantly reduce wound closure time by 30%-225%.^10,16-17

Carney et al¹⁶ demonstrated that within 7 days, thrombin and thrombin peptides can increase incisional wound strength by 55% and 82%, respectively. Over a 7-day period, open wounds closed 39% faster when treated with a thrombin-derived peptide. Substantially improved vascularization also was reported in the thrombin and thrombin peptide–treated wounds.¹⁶

Physiological concentrations of thrombin in wounds can also enhance levels of connective tissue growth factor and vascular endothelial growth factor.^5,9 In tissue culture, thrombin triggers fibroblast constriction of collagen in wound connective tissue matrices.^14-15 Some researchers have postulated that this contraction is part of the process of sealing off the wound during early stages of healing, a wound closure mechanism unrelated to and perhaps more primitive than clot formation. Evidence demonstrates that physiologic concentrations of thrombin or its peptide fragments can increase the rate and quality of wound healing. By inference, antagonism of thrombin’s activities may interfere with wound healing in a variety of ways.

Traditional anticoagulants, agents that exert their preventive action by either direct or antithrombin-mediated inhibition of thrombin, may impair or retard the wound healing process. More selective anticoagulants that slow the rate of thrombin generation without also blocking the activities of this critical enzyme may provide an environment of enhanced protection from thrombosis without interfering with effective wound healing.

Impact of Antithrombotic Activity

Thromboembolic prophylaxis is a critical component of patient management after major joint surgery. Patients undergoing surgery for hip fracture, total hip replacement (THR), or total knee replacement (TKR) are categorized as being at highest risk for development of deep vein thrombosis (DVT) or pulmonary embolism.¹⁸ Without prophylaxis, calf DVT can occur in 40%-80% of patients, whereas proximal DVT can occur in 10%-20% of patients. Pulmonary embolism has been reported in 4%-10% of patients undergoing major joint surgery, and fatal pulmonary embolism can occur in up to 5% of these patients.¹⁸

Venous thromboembolism prophylaxis with pharmacologic agents has become a major component of postoperative management in patients undergoing major joint surgery. Traditional anticoagulants including warfarin, unfractionated heparin, and low-
molecular-weight heparins have been the mainstay of venous thromboembolism prophylaxis. Although effective, these agents do not completely eliminate the risk of venous thromboembolism (Table 1). Newer, more selective anticoagulants have been developed in an attempt to further lower the incidence of venous thromboembolism after major joint surgery. Fondaparinux, a novel synthetic and entirely specific factor Xa inhibitor has demonstrated a significant reduction in venous thromboembolism risk compared with the low-molecular-weight heparin enoxaparin, with no difference in the incidence of clinically relevant bleeding (Table 1).¹⁹ Because most traditional anticoagulants have either a direct or indirect effect on thrombin, beneficial effects on lowering venous thromboembolism must be carefully weighed against a potential adverse impact on the process of wound healing.

Indirect Thrombin Inhibitors

Warfarin

Warfarin acts by interfering with vitamin K–dependent carboxylation of several coagulation factors, including prothrombin (factor II), factor VII, factor IX, factor X, and the anticoagulant proteins C and S.²⁰ Despite being an effective anticoagulant, the use of warfarin in the clinical setting is hindered by numerous limitations, such as unpredictable oral absorption, anticoagulant effect, and modulation of efficacy by other medications. Patient response to warfarin is highly variable, creating major safety concerns. Although the average daily dose to keep patients within the appropriate therapeutic range is 4-5 mg, dosing requirements range from <1 mg per day to >20 mg per day to reach a similar endpoint. Age, hepatic function, underlying disease states, and patient-specific metabolic characteristics influence dosing requirements in patients. Interactions with dietary vitamin K intake, numerous concomitant medications (including herbal and natural products), and lifestyle issues also influence patient response to warfarin.²¹

Warfarin has a narrow therapeutic index, indicating that the margin between efficacy and toxicity is small. Therefore, frequent laboratory monitoring of its therapeutic effect via the international normalized ratio is required to adjust the dose for maximum efficacy without compromising safety; this need for repeated testing detracts from warfarin’s ease of use in clinical practice.^21-22

In addition, due to the slow onset of effect of warfarin, a stable anticoagulant response is usually not achieved until >5 days after the initiation of treatment or any change in dose.²² This delay in reaching full anticoagulant response after initiating warfarin suggests that no immediate effect on thrombin function could adversely impact wound healing. However, the delay in reaching full anticoagulant effect may negatively impact warfarin’s effectiveness in venous thromboembolism prevention. Warfarin instituted within 12-24 hours postoperatively and continued for 2-6 weeks postoperatively is the least effective traditional anticoagulant in preventing venous thrombosis.

Unfractionated Heparin

Unfractionated heparin is a heterogeneous mixture of glycosaminoglycans that was isolated from animal tissue in the early 20th century. Because of the size heterogeneity of heparin preparations (3000-30,000 d), only one-third of the heparin molecules in a given preparation exhibit anticoagulant activity.²³ Unfractionated heparin exerts an anticoagulant effect via interaction with antithrombin. Heparin binds to antithrombin and produces a conformational change in antithrombin, converting it into a rapid inhibitor of thrombin (factor IIa) and factor Xa, as well as several other serine proteases. Most unfractionated heparin preparations inhibit factor IIa and factor Xa in a 1:1 ratio. As unfractionated heparin inhibits both factor Xa and thrombin to the same extent, theoretically the beneficial effects of thrombin on wound healing also may be impacted.

Heparin is heavily sulfated and has a high negative charge density that promotes nonspecific binding to a number of plasma and cellular proteins. This results in decreased bioavailability and substantial interpatient variability in anticoagulant response, and an increased potential for bleeding and thrombotic complications due to heparin-platelet interactions. Therefore, when given in therapeutic doses, unfractionated heparin requires frequent laboratory monitoring to assess the level of anticoagulation, as measured by activated partial thromboplastin time. Similarly to warfarin, heparin requires frequent dosage adjustments limiting its ease of use in clinical practice.²³

Low-Molecular-Weight Heparin

The development of low-molecular-weight heparin resulted from the discovery that shorter heparin chains (3800-5000 d) were sufficient to enhance the anti-factor Xa activity of antithrombin. Low-molecular-weight heparins are derived by the chemical or enzymatic depolymerization of unfractionated heparin and inactivate thrombin to a lesser extent than unfractionated heparin because the smaller molecular fragments cannot simultaneously bind thrombin and antithrombin.

Therefore, low-molecular-weight heparins have an enhanced affinity for inhibiting factor Xa compared to their activity against thrombin. They inhibit factor Xa and thrombin at varying levels ranging in ratio from 4:1 to 2:1.²³ Because low-molecular-weight heparins exert inhibitory effect against thrombin simultaneously with an inhibitory effect against factor Xa, as in the case of unfractionated heparin, there may be a detrimental effect on thrombin’s wound healing properties.

Low-molecular-weight heparins have certain advantages over unfractionated heparin such as better bioavailability, longer half-life, lesser extent of binding to plasma and cellular proteins, and a more predictable dose response. Therefore, monitoring of the intensity of anticoagulation and dose adjustments are not usually required.²³

Factor Xa Inhibitors

Specific molecular targets within the coagulation system have been explored to develop anticoagulant therapies with improved effectiveness, safety, and ease of use. Significant progress has recently been made in the development of factor Xa inhibitors. Fondaparinux is a synthetic version of the pentasaccharide sequence of unfractionated heparin and low- molecular-weight heparin that binds to antithrombin and modifies its conformation, selectively inhibiting factor Xa.²⁴ Because of its short chain (5 sugar units), fondaparinux is unable to directly inhibit thrombin via the antithrombin complex. Therefore, fondaparinux in prophylactic dosage reduces the amount of thrombin generated but also allows a minimal level of thrombin to be formed, which may support coagulation at the surgical wound site. Thrombin formed in this fashion is left undisturbed in the wound and is free to initiate its myriad of healing activities.

Fondaparinux does not require anticoagulation monitoring due to its predictable pharmacokinetic profile and stable dose response. Its half-life of 17-21 hours allows once-daily administration.²⁴ Unlike the heparins, fondaparinux does not affect platelet function, nor does it inhibit platelet aggregation stimulated by a variety of agonists.^25-26

Direct Thrombin Inhibitors

Thrombin is the central effector of coagulation and amplifies its own production, and therefore has been a natural target for direct pharmacologic intervention. Several agents that directly inhibit thrombin (eg, hirudins, bivalirudin, and argatroban) are in clinical use. The agent most widely studied in orthopedic surgery is desirudin. Direct thrombin inhibitors do not require a cofactor, binding the thrombin molecule directly and inhibiting its activity. Unlike the heparins, direct thrombin inhibitors inhibit not only free-floating soluble thrombin but also fibrin-bound or clot-bound thrombin, thus preventing the dual processes of thrombus initiation and propagation.²⁷

Inhibition of thrombin by hirudin significantly reduces the amount of vascular endothelial growth factor released in the wound.⁶ Hirudin also blocks procollagen production by fibroblasts, a process stimulated by thrombin that leads to more rapid tissue repair.¹² Although data on these effects are still sparse, it appears that direct thrombin inhibitors may neutralize thrombin’s beneficial effects in the wound repair process.

Clinical Considerations in Wound Assessment

Any delay in healing of the surgical wound created for implanting a total joint replacement constitutes a major clinical concern. Maintenance of a sterile environment surrounding the implanted artificial joint is a priority. Orthopedic surgeons must carefully balance the use of anticoagulants for venous thromboembolism prophylaxis with the concern of wound bleeding that may lead to complications and delays in the wound healing process. In addition to bleeding in the wound, the seeping of clear exudate is also a cause for concern after major joint replacement, as it may predispose to infection and increase length of hospital stay.

Differences in wound appearance may be related in part to thrombin activity. Direct thrombin inhibitors have been demonstrated to reverse the beneficial effects of thrombin on wound healing.^6,12 Topical thrombin decreases wound hematoma in surgical patients anticoagulated with low-dose unfractionated heparin, further linking the beneficial effects of thrombin to wound healing.^28-29

Clinical studies evaluating the effect of various anticoagulants on wound healing are limited. A recent study presented at the 70th American Academy of Orthopaedic Surgeons meeting evaluated 2001 patients undergoing primary unilateral TKA for postoperative drain output and the time to a dry postoperative wound. The method of DVT prophylaxis did not affect the degree of postoperative drain output. However, the analysis of time to a dry postoperative wound demonstrated that prophylaxis with low-molecular-weight heparin resulted in a significantly greater number of patients with actively draining wounds early in the postoperative period. This difference disappeared by the eighth postoperative day. In addition, low-molecular-weight heparin was associated with a prolonged time to a dry postoperative wound.³⁰

Another study evaluated 205 patients with hip fracture; 114 patients received prophylaxis with low- molecular-weight heparin and 91 received mechanical prophylaxis. Wound status was assessed using the additional treatment, serous discharge, erythema, purulent exudate, separation of deep tissues, isolation of bacteria, and duration of inpatient surgery (ASEPSIS) score. Nineteen percent of patients treated with low-molecular-weight heparin developed wound infection (6% minor, 5% moderate, and 8% severe) compared to 8% of patients on mechanical prophylaxis who developed wound infection (4% minor, 3% moderate, and 1% severe). The incidence and severity of infections were higher in the group that received low-molecular-weight heparin prophylaxis.³¹

These reports raise concern with regard to the most appropriate prophylactic measures after major joint surgery. Future clinical studies are needed to evaluate the effect on wound healing of newer anticoagulants and to evaluate differences between the various anticoagulant agents used for venous thromboembolism prophylaxis.

Conclusion

Anticoagulants used for venous thromboembolism prophylaxis after major joint surgery may have important effects on wound appearance and repair. These effects are likely related to their mechanism of action and activity against thrombin. Orthopedic surgeons must carefully balance the choice and efficacy of anticoagulants with the concern of wound bleeding.

Published data are limited, making the incorporation of wound healing outcome assessments into future large clinical trials of venous thromboembolism prevention efficacy important. Carefully collected prospective data from clinical centers adhering to venous thromboembolism prevention guidelines may also be helpful in generating assessments of anticoagulant effects on wound healing. Given the enormous projected magnitude of hip fracture and hip and knee replacement surgeries in the next decade, evaluating and balancing the specific effects of various anticoagulants on wound healing versus venous thromboembolism prevention efficacy in patients undergoing major orthopedic surgery will be of great importance.

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Authors

From the *Presbyterian Hospital of Dallas, Dallas, Tex; the †University of Illinois at Chicago, College of Pharmacy, Chicago, Ill; and ‡Wayne State University School of Medicine, Harper University Hospital, Karmanos Cancer Institute, Detroit, Mich.

The authors thank David Gibson, PhD, for his advice and generous assistance with the content of this manuscript.