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Inhibitors of Fibrinolytic Components Play Different Roles in the Formation and Removal of Arterial Thrombus in Mice

Matsuno, Hiroyuki *†; Kozawa, Osamu *; Okada, Kiyotaka ; Ueshima, Shigeru ; Matsuo, Osamu ; Uematsu, Toshihiko *

Journal of Cardiovascular Pharmacology: February 2002 - Volume 39 - Issue 2 - p 278-286
Original Articles
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Plasminogen activator inhibitor-1 (PAI-1) and α2-anti-plasmin (α2-AP) may contribute to arterial thrombolysis resistance. The role of these components on thrombus evolution in vivo was investigated in mice deficient for PAI-1 (PAI-1 −/− ) or α2-AP (α2-AP −/− ) or their wild-type counterparts (PAI-1 +/+, α2-AP +/+ ). Moreover, the influence of either PAI-1 or α2-AP deficiency on the results of pharmacologic inhibition of glycoprotein IIb/IIIa of platelets or thrombin was also investigated. A thrombus was induced in the murine carotid artery by endothelial injury. The α2-AP −/− mice were indistinguishable from wild-type, whereas the time to occlusion in PAI-1 −/− was significantly prolonged to 24.9 ± 3.7 min. Vascular patency was markedly increased in both PAI-1- and α2-AP-deficient mice. In separate animals, either a glycoprotein IIb/IIIa antagonist or a thrombin inhibitor was applied. The time required to occlusion was prolonged in a dose-dependent manner in all types of mice. When each compound was administered to PAI-1 −/− mice, significant changes were observed. In conclusion, lack of PAI-1 prolongs the time to occlusion and accelerates clot lysis, whereas α2-AP only has an effect on spontaneous reperfusion. Consequently, the inhibition of PAI-1, but not of α2-AP, could enhance the effects of anti-thrombotic therapy.

*Department of Pharmacology, Gifu University School of Medicine, Gifu, Japan, and †Department of Physiology, Kinki University School of Medicine, Osakasayama City, Japan

Received February 9, 2001; revision accepted August 24, 2001.

Address correspondence and reprint requests to Dr. H. Matsuno at Department of Pharmacology, Gifu University, School of Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan. E-mail: leuven@cc.gifu-u.ac.jp.

Currently available anti-thrombotic therapy includes the use of anti-platelet, anti-coagulant, or thrombolytic agents. For life-threatening acute thrombotic syndromes, e.g., acute myocardial infarction, stroke, or pulmonary embolism, short-term administration of a thrombolytic agent in combination with anti-platelet or anti-coagulant agents is an effective mode of therapy, although recurrent vascular thrombosis following successful thrombolytic therapy remains an important problem (1). The rate and extent of thrombolysis after vascular injury can vary considerably among patients; the molecular determinants responsible for this variation in the lysis of arterial thrombi, which are characterized by high platelet content, are not well understood. The fibrinolytic system contains a proenzyme plasminogen that is converted to the active serine protease plasmin by tissue-type (tPA) or urokinase-type plasminogen activator (uPA). Inhibition of the system may occur through neutralization of the plasminogen activators or through neutralization of plasmin. These neutralizations are achieved mainly by plasminogen activator inhibitor-1 (PAI-1) and α2-anti-plasmin (α2-AP), respectively.

PAI-1, the primary endogenous inhibitor of tPA or uPA, may play a significant role in inhibiting arterial clot lysis (2). Several studies suggest that PAI-1, which inhibits fibrinolysis by binding irreversibly to the active site of tPA or uPA, is a major determinant of the resistance of platelet-rich clots to lysis by tPA (3–5). Indeed, platelets and arterial thrombi contain high concentrations of PAI-1 (6,7), and antibodies to PAI-1 accelerate clot lysis in vitro (8). In vivo, PAI-1-deficient mice are characterized by a reduced thrombus formation (9) and enhanced arterial thrombolysis (10). Conversely, α2-AP is synthesized in the liver and is the main physiologic plasmin inhibitor in plasma, whereas excess plasmin also may become inhibited by α2-macroglobulin (11). Human and murine α2-AP is serine protease inhibitors, which inhibit plasmin in a very rapid reaction resulting in the formation of a stable inactive complex (12). α2-AP levels may be significantly reduced in patients undergoing thrombolytic therapy, especially with non–fibrin specific agents, as a result of extensive systemic generation of plasmin (13). After exhaustion of plasma α2-AP, excess plasmin may degrade several plasma proteins, including fibrinogen, and eventually can lead to bleeding complications. Pathophysiologic observations in humans thus support the relevant role of α2-AP in regulating and controlling plasmin activity.

We previously reported a simple and reproducible thrombus model in the rat femoral artery in which a thrombus is induced by endothelial denudation using a photochemical reaction between rose bengal and green light (540 nm). Direct injury to vessels, such as a strict physical damage by a balloon catheter, was not observed with this application, but a platelet-rich thrombus was induced by endothelial damage without damage to medial smooth muscle cells (14–16). Recently, we applied our system for the production of a carotid artery thrombus in mice, and we investigated the role of the activators of the fibrinolytic system on thrombus formation by using gene-targeted mice with either tPA or uPA deficiency (9). Additionally, we investigated the involvement of tPA or uPA in the inhibition of thrombus formation by a platelet glycoprotein IIb/IIIa antagonist or a thrombin inhibitor (17). Our previous data clearly indicated that tPA plays a more important role than uPA in thrombus formation and that tPA deficiency markedly decreased the effect of a glycoprotein IIb/IIIa blocker (9,18). In the current study, we investigated the role of inhibitors of fibrinolysis on thrombus formation by using gene-inactivated mice with deficiencies of PAI-1 or α2-AP and studied the interaction between the anti-thrombotic drugs, such as a glycoprotein IIb/IIIa antagonist and a thrombin inhibitor, and either PAI-1 or α2-AP deficiency on thrombus formation.

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METHODS

Animals

Deficient mice were generated by homologous recombination in embryonic stem cells, as described previously (19–20). Unless otherwise stated, the backgrounds of all mice were mixed C57BL/6 and 129 (H-2 b ). All these studies were performed on young adult mice (3–5 months of age). All experiments were performed in accordance with institutional guidelines.

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Reagents

GR144053, a platelet membrane glycoprotein IIb/IIIa antagonist, and argatroban, a thrombin inhibitor, were kind gifts from Glaxo Wellcome, Middlesex, U.K. and Mitsubishi Chemical Co., Ltd. (Yokohama, Japan), respectively. Collagen for platelet aggregation was obtained from Nycomed Arzneimittel GmbH (Munich, Germany). The other chemical substances were obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, U.S.A.).

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Production of endothelial injury

The experimental procedure to induce an endothelial injury has been described in detail previously (17). Mice (n = 8, each) were anesthetized by i.p. injection of 44-mg/kg sodium pentobarbital. In brief, the right common carotid artery, the left jugular vein, and the right femoral artery were exposed under the anesthesia with pentobarbital. Catheters (internal diameter [ID] D = 0.5 mm, outer diameter [OD] = 0.8 mm, polyethylene sp3, Natume Co., Ltd., Tokyo, Japan) were connected to the left jugular vein and to the right femoral artery for the injection of rose bengal (30 mg/kg) and for monitoring of blood pressure and pulse rate using a pressure transducer (AP601G Nihon Koden, Tokyo, Japan) during experiments on day 0. Blood flow in the carotid artery was continuously monitored using a Doppler flow probe (Model PDV-20, Crystal Biotech Co., Ltd., Tokyo, Japan) positioned distally to the injured area of the carotid artery. Irradiation by green light (540 nm) proximal to the flow probe was started and then rose bengal was injected as a bolus 10 min after the observation of control blood flow. The irradiation was continued for 15 min after the injection of rose bengal. This procedure results in destruction of endothelial cells in the irradiated area by oxygen radicals induced by the photochemical reaction between rose bengal and green light. Our previous histologic observations revealed that a platelet-rich thrombus including fibrin was formed when the blood flow was 0 (17). An occlusive thrombus formation was judged to occur when blood flow was 0. After recovery from anesthesia, the animals were kept in individual cages and fed standard chow (RC4, Oryental Yeast Co., Ltd., Tokyo, Japan). The mice were reanesthetized and carotid blood flow was measured on each observation day after injury.

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Infusion regimen to prevent in vivo thrombus formation

In separate experiments (n = 8, each group), GR144053 or argatroban was administered by continuous i.v. using an infusion pump (Termo STC-523, Termo, Tokyo, Japan). The infusions were started 20 min before the initiation of endothelial injury and continued for 90 min thereafter. Animals were divided into a control group (saline infusion), a group treated with GR144053 (0.01, 0.03, 0.1, 0.3, 1.0, or 3.0 mg/kg/h), and a group treated with argatroban (0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 mg/kg/h).

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Bleeding times

In the dose-dependent studies of animals treated with GR144053 or argatroban (n = 4, each group), bleeding time measurements were performed as described (21). A distal 2-mm segment of tail was severed with a razor blade at the end of the infusion period. The tail was immediately immersed in 0.9% saline at 37.0°C with the tip of the tail 5 cm below the body. The bleeding time was defined as the time required for cessation of blood flow (blood did not spread in saline).

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Ex vivo platelet aggregation

Blood was collected by heart puncture on sodium citrate (3.15%) and centrifuged for 10 min at 155 g to obtain platelet-rich plasma after the end of infusion of GR144053 or argatroban in dose-dependent studies (n = 4, each group). Platelet aggregation was induced by collagen and followed in an aggregometer (Aggrecorder II, DA-3220, Kyotodaiichi-Chemical, Kyoto, Japan) at 37°C with a stirring speed of 800 rpm. Aggregation is expressed as percentage of maximum light transmission obtained in the absence of drugs. All measurements were done in duplicate.

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Anti-coagulant studies ex vivo

After the separation of platelet-rich plasma for platelet aggregation, remaining blood samples were further centrifuged for 10 min at 1,550 g to obtain platelet poor plasma. The thrombin time was determined using standard clinical laboratory procedures (22).

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Statistics

All data are expressed as mean ± SEM. The significance versus data from the wild-type mouse was determined by analysis of variance followed by the Wilcoxon test for the time to occlusion in vivo. For vascular patency after spontaneous reperfusion, the significance versus the wild-type mouse was determined by the χ 2 test.

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RESULTS

Acute thrombus formation

The times to occlusion after injury of the carotid artery in each group are shown in Figure 1. Blood flow in the carotid artery of wild-type mice stopped after 11.3 ± 1.1 min (PAI-1 +/+ ) and 11.9 ± 0.8 min (α2-AP +/+ ). In α2-AP −/− mice, the time to occlusion was slightly but not significantly prolonged to 14.1 ± 3.1 min. In PAI-1 −/− mice, however, the time to occlusion was significantly prolonged to 24.9 ± 3.7 min (p < 0.05 versus PAI-1 +/+ mice).

FIG. 1.

FIG. 1.

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Spontaneous thrombolysis and vascular patency after reperfusion

The time profiles of vascular patency in each group after the initiation of endothelial injury by the photochemical reaction are illustrated in Figure 2. Spontaneous reperfusion was observed in five of 16 wild-type mice at the end of the observation period on day 0, which was consistently associated with cyclic reocclusion and reflow. On day 1, persistent occlusion was observed in two mice, cyclic reocclusion and reflow in 12, and persistent patency in two mice. On day 2, cyclic reocclusion and reflow was observed in eight mice and the others showed persistent patency. On day 3, all arteries in the wild-type mice remained patent. Spontaneous reperfusion was clearly observed in all arteries of PAI-1 −/− mice within 90 min on day 0; however, with a lot of cyclic reocclusion/reflow. The arterial patency status of α2-AP −/− mice was similar to that of wild-type mice on day 0, but spontaneous reperfusion on day 1 was observed in all mice (Table 1).

TABLE 1

TABLE 1

FIG. 2.

FIG. 2.

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Anti-thrombotic effect of GR144053 and argatroban in vivo

In the wild-type control mice, the times to occlusion were prolonged by treatment with either GR144053 or argatroban in a dose-dependent manner (Figs. 3 and 4). Administration of GR144053 at a dose of 0.1 mg/kg per hour did not significantly change the time required to occlude the carotid artery. However, ≥1.0 mg/kg/h resulted in a significant prolongation, whereas on treatment with 3.0 mg/kg, five of the 12 arteries examined no longer occluded within the observation period, although the blood flow was decreased. In contrast, when GR144053 was given to PAI-1 −/− mice, a dose of only 0.1 mg/kg/h resulted in a significant prolongation of the time to occlusion. Moreover, 0.3 mg/kg/h of GR144053 was able to prevent occlusion of all arteries during the observation period. The estimated median effective dose (ED 50 ) for GR144053 in PAI-1 −/− mice is 0.064 mg/kg/h, which is 11.4 times lower than the one for wild-type mice (ED 50 = 0.73 mg/kg/h). GR144053 had a similar inhibitory effect on the development of a thrombus in α2-AP −/− and wild-type mice. GR144053 at a dose of 1.0 mg/kg/h significantly prolonged the time to occlusion. The estimated ED 50 in α2-AP −/− mice (ED 50 = 0.84 mg/kg/h) was slightly decreased but was not significantly different from the controls (ED 50 = 0.90 mg/kg/h).

FIG. 3.

FIG. 3.

FIG. 4.

FIG. 4.

Conversely, argatroban had a similar inhibitory effect against the development of a thrombus. When argatroban was given to PAI-1 −/− mice (ED 50 = 0.22 mg/kg/h), the estimated ED 50 was significantly decreased as compared with that of wild-type mice (ED 50 = 1.68 mg/kg/h). On the contrary, the estimated ED 50 for argatroban in α2-AP −/− mice (ED 50 = 1.61 mg/kg/h) was not different as compared with that of α2-AP +/+ mice (ED 50 = 1.72 mg/kg/h).

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Bleeding time and hemostasis analysis

Bleeding time is given in Table 2. No significant differences were observed between gene-deficient and wild-type mice. When a 2-mm segment of tail was amputated in PAI-1 −/− or α2-AP −/−, bleeding time was slightly prolonged as compared with wild-type mice. Conversely, when GR144053 or argatroban was administered, the bleeding time was prolonged in a dose-dependent manner in all types of mice. In particular, the bleeding time was significantly prolonged when the highest dose of GR144053 (3.0 mg/kg/h) or argatroban (3.0 mg/kg/h) was administered to deficient mice.

TABLE 2

TABLE 2

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Inhibition of platelet aggregation ex vivo

Maximum aggregation obtained with platelets from groups treated with GR144053 is shown in Figure 4. The changes from the control (infusion of saline) value were statistically significant when GR144053 was given at doses > 0.3 mg/kg/h in all types of mice. The estimated median inhibitory concentration values in all mice types were not significantly different. Argatroban did not affect platelet aggregation induced by collagen.

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Anti-coagulant effects

GR144053 had no significant effect on coagulation parameters. Argatroban prolonged thrombin time dose dependently in all types of mice, with a significant prolongation at the highest dose of argatroban used (Fig. 5).

FIG. 5.

FIG. 5.

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DISCUSSION

The present study was performed to evaluate the role of inhibitors of fibrinolytic system components on arterial thrombus formation in vivo by using mice deficient in either PAI-1 or α2-AP. Moreover, the interaction between these fibrinolytic components and anti-thrombotic agents on the thrombus formation was investigated.

According to the results of our studies in wild-type mice, we are able to induce a moderate thrombus formation in the carotid artery, because 31.5% of the occluded arteries spontaneously reperfused at the end of the observation period on day 0, despite the observation that the first complete thrombotic occlusion had already occurred within 15 min in all arteries. On the third day, all arteries were persistently patent. Spontaneous reperfusion after vascular occlusion is an important event in acute ventricular remodeling after myocardial infarction (23) and in ischemic stroke (24). Our previous data showed that lack of tPA significantly affected spontaneous reperfusion after the development of thrombus (18). In this study, PAI-1 deficiency affected both the development and lysis of the thrombus. Several in vitro studies suggested that PAI-1 is the dominant factor in inhibiting clot lysis (3,4); however, other studies indicated that it has only a marginal role (25). These studies, however, did not take into account the role of the vascular wall in the regulation of thrombolysis, because, for instance, the role of PAI-1 in regulating thrombolysis in vivo involved the injection of preformed venous clots or of whole blood mixtures into isolated arterial segments (26,27). Our experiments demonstrated that in mice lacking PAI-1, significantly more time is required to form an occlusive thrombus, and that vascular patency by spontaneous reperfusion is accelerated.

Conversely, neither the time needed for the formation of an occlusive thrombus after endothelial injury nor the patency status on day 0 was significantly affected by the lack of α2-AP: five of eight arteries were occluded at the end of the observation period even though cyclic flow reduction was observed in all arteries. However, arterial patency status in α2-AP −/− mice on day 1 was markedly changed and was similar to that seen in PAI-1 −/− mice. These results indicate that α2-AP mainly plays a significant role in the events of vascular reperfusion and the reduction of reocclusion but not in the prevention of occlusive thrombus. Indeed, clot lysis experiments confirmed that the endogenous thrombolysis potential is significantly enhanced in α2-AP −/− mice, indicating a physiologic role of α2-AP −/− in fibrin surveillance (27). Recently, lack of α2-AP significantly decreased the size of thrombus in mice when endothelial injury was induced by photochemical reaction between rose bengal and green light (28). However, in our experiments, the condition of endothelial injury by photochemical reaction was much more severe and lack of α2-AP could not fulfill its function for prevention of thrombus formation. Additionally, Kawasaki et al. (28) finally concluded that vessel wall PAI-1 plays a major role in thrombus stabilization.

The combined treatment with different types of anti-thrombotic and thrombolytic drugs could have significant effects on vascular occlusion by thrombus formation. Therefore, we investigated the interaction between fibrinolytic inhibitors and anti-thrombotic compounds using mice deficient in PAI-1 or α2-AP. Temporary prevention of glycoprotein IIb/IIIa availability, representing the final step in platelet aggregation, is expected to decrease thrombus formation resulting from multiple proaggregatory platelet stimuli. Indeed, the presence of a glycoprotein IIb/IIIa antagonist decreased the time required to attain vascular reperfusion by thrombolytic drugs and subsequently maintained the arterial blood flow (29). Conversely, the inhibition of thrombin function results in the reduction of fibrin formation and has an inhibitory activity on platelet aggregation induced by thrombin in vivo because thrombin is a strong platelet activator. In this study, GR144053 or argatroban had a significant anti-thrombotic effect in wild-type, PAI-1 −/−, and α2-AP −/− mice. However, the strongest effects were observed with PAI-1-deficient mice, in which the time to thrombotic occlusion was markedly prolonged as compared with the other types of mice. Indeed, the potential anti-thrombotic effect of an inhibitor of PAI-1 has been indicated by both in vitro and in vivo experiments (30). An inhibitory monoclonal antibody against PAI-1 inhibited thrombus growth and enhanced clot lysis in a rabbit model of venous thrombosis (31). In our results, the lack of PAI-1 markedly enhanced the inhibitory effect of either a glycoprotein IIb/IIIa antagonist or a thrombin inhibitor on thrombus formation without a significant hemorrhagic risk. PAI-1 is abundant in platelets, from which it can be secreted, and also can be also secreted by vascular endothelial cells when stimulated, for instance, by factors released from activated platelets (32). These observations showed that the anti-thrombotic effect obtained by the combined inhibition of PAI-1 and either platelet glycoprotein IIb/IIIa or thrombin may make a powerful mix to prevent vascular occlusion in vivo. Conversely, α2-AP also plays a role in the suppression of the fibrinolytic system, but our data indicate that the lack of α2-AP does not modulate the efficacy of the anti-thrombotic therapy by an agent blocking glycoprotein IIb/IIIa or inhibiting thrombin in vivo. From these data we conclude that PAI-1 plays a more prominent role in thrombosis-mediated vascular occlusion than α2-AP.

It should be noted that anti-thrombotic interventions might be easily complicated by hemorrhagic events. In our experiments, the anti-thrombotic effects of either GR144053 or argatroban were paralleled by a prolongation of the bleeding time. The bleeding tendency was more pronounced in PAI-1- or α2-AP-deficient mice when the anti-thrombotic compound was given. However, the dose of GR144053 or argatroban preventing vascular occlusion by thrombus in PAI-1 −/− mice did not prolong the bleeding time. These findings indicate that, under the condition of lack of PAI-1, it would be easy to achieve a sufficient anti-thrombotic effect by the inhibition of glycoprotein IIb/IIIa or thrombin without bleeding risk. We speculated about the difference between hemorrhage risk and anti-thrombotic effect in PAI −/− mice combined with an anti-thrombotic compound as follows: In the process of thrombus formation in vivo, damage of endothelial cells is a trigger for the development of thrombus. When endothelial cells are stimulated by physiologic injury or shear stress, secreted tPA and PAI-1 from endothelial cells could regulate in the fibrinolytic system in different directions. Conversely, hemorrhagic events are related to not only endothelial cell dysfunction but also to other physiologic factors, such as the function of platelets and of coagulation factors. Namely, PAI-1 deficiency plays a key role in the prevention of thrombus formation in vivo. On the contrary, the combination of the lack of α2-AP with the presence of an inhibition of glycoprotein IIb/IIIa or thrombin results in a significant risk for hemorrhagic events when aiming to obtain a sufficient anti-thrombotic effect.

We therefore speculate that the physiological response to injury might be as follows: First, after endothelial injury, platelets adhere and aggregate on the damaged endothelial area, after which the fibrin net forms and strengthens the platelet-rich thrombus. During this process, mainly PAI-1, and to a much lesser extent α2-AP, limit the activity of fibrinolytic components. Locally secreted tPA by endothelial cells at the edges of the injury area, or uPA in circulation, would limit the growth of the thrombus to the region of injury and would dissolve any thrombus that would extend beyond the boundaries of the injury. This is in line with our observations that spontaneous reperfusion of occluded vessels is facilitated in PAI-1 −/− mice but reduced in tPA −/− mice (18). These findings suggest that in particular inhibition of PAI-1 activity and secretion may further enhance the anti-thrombotic effect of anti-thrombotic compounds because the occlusive thrombus is composed of both activated platelets and fibrin. Conversely, the inhibition of fibrinolysis by α2-AP would likely have its main role in the circulation but not in the injured endothelial surface on the development of thrombus formation, because a lack of α2-AP clearly improves vascular patency after spontaneous reperfusion.

In conclusion, our findings indicate that PAI-1 plays a significant role not only in the development of thrombus but also in spontaneous reperfusion or reocclusion. Moreover, lack of PAI-1 enhances the anti-thrombotic effect obtained by inhibiting platelet glycoprotein IIb/IIIa or thrombin. On the contrary, α2-AP deficiency would mainly facilitate the lysis after the establishment of thrombus but would not affect the development of thrombus. These findings could be useful for further clinical development of anti-thrombotic therapy.

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Keywords:

α2-Anti-plasmin; Gene-inactived mice; Plasminogen activator inhibitor-1; Reperfusion; Thrombus

© 2002 Lippincott Williams & Wilkins, Inc.