An experimental study on the use of an antagonist of the platelet glycoprotein IIb/IIIa receptor in the prevention of thrombosis in vascular microanastomoses

Abstracts

O autor comparou, experimentalmente, a eficácia da droga abciximab, um antagonista do receptor da glicoproteína IIb/IIIa das plaquetas, na prevenção da trombose nas microanastomoses arteriais em ratos Wistar. Utilizou 20 animais, dos quais 10, do grupo controle, receberam solução salina e 10 receberam abciximab, na concentração de 0,8mg/kg, injetados na veia femoral. Trinta minutos após a administração da solução salina ou abciximab, todos os animais foram submetidos ao mecanismo provocador de trombose vascular da artéria femoral do lado oposto ao utilizado para administração da droga, por meio de um trauma externo promovido por aparelho IMPACTOR, desenvolvido pela New York University para padronização de lesão da medula nervosa, o que padronizou a lesão arterial. Todos os animais foram submetidos à microanastomose vascular com mononáilon 10-0, em pontos separados, no local do trauma externo. Foram realizados testes para analisar a perviabilidade vascular da artéria femoral no período de 10, 20 e 30 minutos após o término da anastomose. Após este período, todas as artérias femorais submetidas às microanastomoses foram ressecadas e analisadas em microscopia óptica para avaliar a presença de formação de trombos. Após a análise estatística dos dados, o autor concluiu que o uso do antagonista do receptor da glicoproteína IIb/IIIa das plaquetas diminuiu a incidência de tromboses nas microanastomoses vasculares.

complexo glicoprotéico GPIIb/IIIa de plaquetas; trombose; microanastomose cirúrgica; artéria femoral; ratos wistar

ARTIGO ORIGINAL

An experimental study on the use of an antagonist of the platelet glycoprotein IIb/IIIa receptor in the prevention of thrombosis in vascular microanastomoses

Márcio Issamu Oide

SUMMARY

The author has experimentally compared the efficacy of the platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) in the prevention of platelet aggregation and thrombosis in microvascular surgery using rat models. Twenty rats were used: ten rats were used as a control group and received saline; tem rats received abciximab 0.8 mg/kg administered in the femoral vein. After thirty minutes following administration, all rats underwent vascular thrombosis of the opposite femoral artery through an external trauma promoted by IMPACTOR, a device developed by the New York University to serve as a model of the spinal cord injury and the arterial damage. All rats were submitted to microvascular anastomoses using mononylon 10-0 and separated suture at the site of the external trauma. Patency tests were performed on femoral arteries 10, 20 and 30 minutes after microanastomoses. All femoral arteries were removed for pathological studies to detect presence or absence of thrombus generation at the microanastomoses. After statistical analyses, the author concluded that the use of the platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) avoided platelet aggregation and thrombosis in microvascular surgery.

Key words: platelet glycoprotein IIb/IIIa complex / drugs effect; thrombosis / prevention, microvascular surgery; femoral artery; Wistar rats.

INTRODUCTION

The vascular microsurgery has advanced over the last four decades, since its introduction by JACOBSON and SUAREZ, in 1960. The progress was due either to the improvement of microsurgical devices, optical instruments and microsurgical wires, or the improvement in surgeons training. As a result, there was a decline in the incidence of microanastomoses thrombosis rates, although it still represents one of the main causes of failures in the microsurgical reconstructions.

For preventing thromboses, antithrombotic agents like heparin, antiplatelets like acetylsalicylic acid, and glicoprotein IIb/IIIa receptor antagonists have been used.

Heparin inhibits thrombin indirectly thanks to its reversible binding with the anticoagulant named antithrombin III. Antithrombin III is a natural inhibitor of the activated clotting factors (ROSENBERG; ROSENBERG 1984 and SALZMAN et al. 1980). The complex heparin-antithrombin III enhances the effect of antithrombin through the inactivation of thrombin and clotting factors as XIIa, XIa, IXa and Xa.

The acetylsalicylic acid, associated or not to dipiridamole, may reduce thrombi incidence and formation significantly (BARNATHAN et al. 1987 and FUSTER et al. 1993). However its efficacy as an antiplatelet agent is low. The acetylsalicylic acid inhibits the thromboxane A2 generation from platelets and the subsequent activation of other platelets thanks to the irreversible blockade of the cyclooxigenase enzyme (JANG et al. 1992).

Although thrombin is a potent agonist of the platelet aggregation, either heparin or acetylsalicylic acid is weak antiplatelet agents. They have a limited activity because other agonists as collagen, adenosine diphosphate, adrenaline and serotonin can activate platelets aside from thrombin and the cyclooxigenase pathway, by activating platelet glycoprotein IIb/IIIa receptors (JANG et al. 1992).

In order to avoid thrombosis effectively we should give a number of drugs to block each pathway of the platelet activation or a single substance, like an antagonist of the glycoprotein IIb/IIIa receptor for the fibrinogen, which is present in platelets membranes and is responsible for the platelets aggregation. COLLER et al. (1991) described a new murine monoclonal antibody (7E3) against the fibrinogen receptor of the platelet glycoprotein IIb/IIIa as an antithrombotic agent. This monoclonal antibody 7E3, abciximab, has been widely used in cardiology for preventing heart ischemic complications in high-risk patients underwent percutaneous transluminal coronary angioplasty or arteriotomy (EPIC investigators 1994, LEFKOVITS et al. 1997, ELLIS et al. 1998).

Because we are interested in preventing and decreasing the incidence of complications of the microsurgical procedures related to the thrombosis of vascular microanastomoses, we had the incentive to develop and create an experimental model with the objective of testing new drugs and procedures. In addition, we usually follow the clinical and surgical activities from Institute of Orthopedics of São Paulo University.

In spite of few reports in the literature related to the use of the murine antibody against the platelet glycoprotein IIb/IIIa receptor to avoid thrombosis in vascular microanastomoses (STOCKMANS et al. 1997), we ponder that the use of this drug might modify and reduce the thrombosis incidence.

In order to use the murine monoclonal antibody 7E3 - abciximab - to prevent thrombosis in microsurgical procedures, we decided to test it through an experimental model in mice to evaluate its efficacy.

MATERIAL AND METHODS

A total of twenty Wistar mice were used with a mean weight of 352,37 g (ranging from 260 g - 417g) and aged five months at the enrollment. The mice were selected into two groups of ten - Group I and Group II.

Surgical Technique

The same surgeon performed all surgeries. All animals were anesthetized with ketamine solution (Ketalarâ) 80 mg/kg plus 2% thiazine cloridrate (Rompunâ) 3.2 mg/kg given intraperitoneally. Each animal was lying down in dorsal decubitus. The thighs and the inguinal region were shaven. Antisepsis was made using ethyl alcohol (Figure 1). An incision of 3-cm in the skin of the inguinal region between the abdomen and the thigh was performed. Femoral vascular and nervous structures were individualized and dissected. Then it was identified: the artery (centrally), the vein, (medially) and the nerve (laterally) (Figure 2). Hemostasis was performed through a bipolar electric bistoury with the aid of a surgical microscope. In the lumen of the femoral vein, a polyethylene catheter no. 24G (NIPROâ) was introduced to inject abciximab (ReoProâ) 0.8 mg/kg. Group II was given the drug while Group I was given a saline solution 0.9% (Figure 3). The same procedures were followed for the dissection of the pedicle in the opposite paw. Once the femoral artery was isolated, a plate of plastic material measuring 20 mm x 5 mm x 1 mm was placed under the artery. After 30 minutes of the drug administration, three contusion lesions were made in the femoral artery at 15-second intervals using IMPACTOR device (New York University SPINAL CORD CONTUSION SYSTEM) which left the pendulum fall from a height of 50 cm (Figure 4). After the lesion, the artery was sectioned using microsurgical scissors. Then the vascular microanastomosis by the technique of separate points using nylon thread 10-0 and needle of 75 micra was made. In all microanastomoses the saline solution did not contain heparin (Figure 5 and 6).

After the suture conclusion, the presence or absence of blood flow 30 minutes after reconstruction was observed at each 10 min, through the test of the perviousness with microsurgical tweezers according to PEDERSON; SANDERS (1999) and ACLAND (1981). Femoral arteries underwent vascular microanastomoses were removed and immersed in formaldehyde 10% and forwarded to histological analysis. The histological analysis used hematoxylin-eosine, and Verhoff and Masom technique. Arteries were sectioned obliquely into pieces of 2 mm of thickness, and fixed in formaldehyde 10% for 24 hours. The histological material was immersed in paraffin. An automatic rotative microtome was used for obtaining 5-10 pieces of 5 micra of each artery that were stained. The sheets were microscopically observed for the absence or presence of thrombi. Following this, mice were underwent euthanasia through the administration of 100 mg/kg of sodium pentobarbital intraperitoneally (SOUZA, 1996). The results were assessed through the test of the vessel perviousness using microsurgical tweezers observed at the surgical microscope (PEDERSON; SANDERS, 1999 and ACLAND, 1981) and also through histological analysis of the area of the vascular micro-anastomosis.

Both Groups were statis-tically compared through Fisher's test with 0,05 of significance.

RESULTS

We evaluated the blood flow 10, 20 and 30 minutes after the microanastomoses of the femoral artery, as well as the thrombi incidence in the histological exam.

Chart I

Chart II

Comparing the blood flow in the microanastomosis at 10, 20 and 30 minutes in both Groups, no statistically significant difference is observed (Table1, ² and ³).

Comparing the thrombosis incidence in the microanastomosis noted in the histological exam after 30 minutes in both Groups (Figures 17, 18, 19 and 20), a statistically significant difference was observed (Table 4).

DISCUSSION

Since our initial activities in the Hand Division of the Institute of Orthopedics from Hospital das Clínicas of the School of Medicine of São Paulo University, we have been performing a number of procedures that require arterial and venous microanastomoses. These procedures involve reimplantation and free tissue transference (cutaneous, fascial, muscular, and bony tissues). In spite the success high rates most failures occur due to microanastomoses thrombosis.

Microanastomoses thrombosis is a challenge and a common complication even for skilful surgeons and experts. For its prevention, we have to understand its pathophysiology better.

In the classical ACLAND study (1973) it was emphasized that the non-traumatic microsurgical technique contributes little for preventing the microanastomosis thrombosis. The presence of thrombi rich in platelets in the suture site (vessel walls, vessel incision and vessel incision followed by vessel suture) suggests that thrombogenic factors be related to the endothelial lesion.

Although some experiments suggest that the suture material or the disturbances of the flow in the anastomosed area are the main causes for the development of the thrombosis, DICKSON et al. (1993) and JOHNSON et al. (1992) demonstrated that both are secondary factors. JOHNSON; BARKER (1992) and HILL; WHITTEN (1997) considered that endothelial lesions with subendothelial exposure are responsible for anastomoses thrombogenicity, since the endothelial injury may activate the coagulation cascade and platelets.

Among the anticoagulant substances, heparin acts avoiding thrombin generation. Once thrombin is activated it converts fibrinogen to fibrin. Heparin enhances the activity of antithrombin III, which inactive the activity of thrombin and the clotting factors XIIa, XIa, IXa and Xa. Heparin can be administered either systemically, as described by COOLEY; HANSEN (1985) and GREENBERG et al. (1988), or topically. Its efficacy in thrombi prevention is increased when heparin is added to a saline solution and the vessel is flushed during the procedure of vascular microanastomosis, as demonstrated ZINBERG (1989), COX (1992) and BRAAM (1995). KOROMPILIAS (1997), when studied a heparin of low molecular weight (enoxaparin) reported that there are no benefits in the systemic or topical administration with relationship to the single topical irrigation. Bleeding, hemodynamic disturbs and hematomas are complications of the systemic administration of heparin, and also indirect causes of thrombosis of the vascular microanastomoses.

HUPKENS, COOLEY (1996) compared heparin and antiplatelets efficacy. Heparin is more efficacious in preventing microanastomoses thrombosis. In this trial, the authors analyzed antiplatelets (acetylsalicylic acid and dipiridamole) which do not inhibit the final pathway of the platelet aggregation, i.e., the activation of the platelet glycoprotein IIb/IIIa receptors, but the pathway of the cyclooxigenase. Probably, for this reason, they obtained worse results with antiplatelets.

Glycoproteins are major structural components of the platelets' membrane, which act as receptors. Glycoprotein Ib and glycoprotein from the IIb/IIIa complex are the most recognized.

Glycoprotein Ib has receptors for the von Willebrand clotting factor and thrombin, which fixes to the platelet surface forming a bridge or a connection between the platelet and the subendothelial collagen, which also has receptors for this factor. Von Willebrand factor plus glycoprotein Ib form a channel in the platelet membrane, which allows the influx of calcium ions. These increased calcium levels activate glycoprotein IIb/IIIa, promoting its connection between von Willebrand factor and fibrinogen, and causing platelet aggregation (SAKARIASSEN et al., 1986). Such discoveries represented an important advance in searching for more efficient drugs to inhibit platelet aggregation.

The deficiency of glycoproteins in platelets leads to platelet diseases and bleeding. The most recognized disturbs are Bernard-Soulier syndrome and Thromboasthenia of Glanzmann. Bernard-Soulier syndrome is characterized by platelet and membrane glycoprotein Ib decreases. Platelet adhesion to the harmed endothelium does not occur, which leads to bleeding episodes. In Thromboasthenia of Glanzmann, there is lack of glycoprotein IIb/IIIa receptors, number of platelets is normal, but their aggregation is deficient. In spite of the defective aggregation, platelets have normal adhesion to the harmed vascular subendothelium (SAKARIASSEN et al., 1986). Such diseases helped us to understand the effects of the inhibition of the platelet aggregation by using the monoclonal antibody against glycoproteins IIb/IIIa receptors (COLLER et al., 1983).

The acetylsalicylic acid inhibits the platelet aggregation through the inhibition of the thromboxane A2 synthesis (via cyclooxigenase), which activates the glycoprotein IIb/IIIa receptor. However, other substances like collagen, adenosine diphosphate, adrenaline, thrombin and serotonin may also activate the glycoprotein IIb/IIIa receptor, unchaining the platelet aggregation, even if the cyclooxigenase pathway is blocked. Due to this aspect, we find several reports on the low efficacy of the acetylsalicylic acid in preventing microanastomoses thrombosis (COOLEY; GOULD, 1987 and STOCKMANS et al., 1997).

The discovery of platelet glycoproteins IIb/IIIa receptor antagonists makes possible the receptor blockade, avoiding stimulation by any one of the pathways, and providing larger antiplatelet efficacy. In addition, it avoids the binding with the receptor principal agonist, thrombin.

COLLER et al. (1985,1986) developed the murine monoclonal antibody 7E3, which blocks the binding of fibrinogen with the glycoprotein IIb/IIIa receptor complex. The purified fragment F (ab) ₂ is capable to block 85% of receptor sites, reducing platelet aggregation in more than 90%, even in the presence of agonist substances like adenosine diphosphate, adrenaline, thrombin and collagen. Although the bleeding time is prolonged in more than 30 minutes, that author in experimental studies in dogs did not describe significant bleedings.

BATES et al. (1991) studied the preventive use of 7E3 to avoid thrombosis in coronary angioplasty in dogs, showing its high efficacy. This trial provided the use of the drug as an adjuvant in avoiding acute ischemic complications associated to the high-risk angioplasty, allowing patients to be treated by this percutaneous technique.

In recent studies, LINCOFF et al. (1997), ELLIS et al. (1998), GHAFFARI et al. (1998) and HAASE et al. (1999) demonstrated the platelet glycoprotein IIb/IIIa receptor antagonist (murine monoclonal antibody 7E3 - abciximab) efficacy and low incidence of side effects in patients with unstable angina underwent high-risk coronary angioplasty. Benefits overcome the inherent risks of bleeding.

The objective of this experimental study was to know the efficacy of abciximab for the prevention of thromboses in the vascular microanastomoses. Such a study has not been performed yet, which stimulated us to research the effect of the referred drug in this surgical procedure.

In order to perform the present experimental work Wistar mice were used once they are an experimental model of femoral artery microanastomoses well known (ACLAND, 1981). Twenty male and female animals, aged five months, whose weight ranged from 417 g to 260 g (the mean weight of Group I was 355,8 g and the mean weight of Group II was 292,3 g) were used. Such difference in body weights did not interfere in our results since the administration of the drugs was calculated according to the weight of each animal. In spite of Group I mice were relatively larger than Group II, we did not observe significant differences in relation to the lumen of the vessels to be anastomosed.

The same surgeon to minimize possible technical differences accomplished all microanastomoses.

The anesthesia was accomplished with ketamine 80 mg/kg plus 2% thiazine cloridrate 3.2 mg/kg, intraperitoneally. These drugs are safer than pentobarbital, once the risk of respiratory depression is lower. The anesthetic effect lasts 90 minutes on average, which is enough time to perform our studies. The association of drugs provided to use more anesthetics (50% of the first dose) with no risk of respiratory complications and death of the animal.

In our experimental model, the femoral artery was chosen due to the easier access and lesser morbidity for animals comparing to other vessels, like the carotid artery.

The femoral vein of one of the animal's paw was dissected. Animals of Group II and Group I was given either the drug or saline solution intravenously, respectively. The opposite paw was used for performing microanastomoses.

According to COLLER, SCUDDER (1985), the concentrations used in mice (0.8 mg/kg) were the same ones used in dogs. In the literature, we did not find any report referring to the optimal concentration to be applied in mice to block the glycoprotein IIb/IIIa receptors effectively. AHN et al. (1999) uses abciximab 2 mg/kg.

In the initial project, the biomechanical laboratory and us made a "clamp" to promote the arterial injury. However, we could not pattern lesions because it as difficulty to determine the pressure which was applied, as well as the minimum time to determine the arterial injury.

In this study, the main objective of using Impactor New York University was patterning the artery injury, creating a new experimental model for thrombosis. We accomplished three subsequent traumas, which is the minimum to promote thrombi generation in most of the arteries of the mice. The device used was developed to pattern the bone marrow lesion in mice. According to RODRIGUES (1999), when a pendulum falls from 50 cm of height it determines bone marrow injury effectively. Thus, this height is adequate to provide the arterial lesion.

During the arterial microanastomosis, heparin was not used in the irrigation, because it could inhibit thrombi generation. BRAAM (1995) and COX (1992) emphasized such aspect.

In clinical practice, vessel lesions occur even after the administration of antithrombogenic drugs or simultaneously to microanastomoses. In this experimental trial, the main objective of the altered sequence (administration of the drug - observation for 30 minutes - arterial lesion promotion _ anastomosis) was to provide antibodies to bind to platelet glycoprotein IIb/IIIa receptors (BATES et al., 1991). On the other hand, the experimental model for arterial lesion was so effective that, soon after the pendulum fall, and before the microanastomosis, the thrombosis occurred within 30 minutes when abciximab was not given.

We analyzed the flow presence and the maintenance of perviousness of the artery every 10 minutes for 30 minutes. According to BOECKX (1994) and BATES (1991) after the microanastomosis, the first 30 minutes are critical for thrombus generation. Thus, 30 minutes after anastomoses, we removed the femoral arteries and performed animals' euthanasia.

The efficacy of abciximab is 80% for 120 minutes following the administration (TCHENG et al., 1994). To maintain its efficacy for a larger period, the drug should be administered as an intravenous continuous infusion for 24 hours, which is not viable in mice.

The fragment of the femoral artery that was removed included the proximal and the distal segment to the suture, which are critical areas for thrombi generation. The fragments underwent histological analyses at Institute of Orthopedics and Traumatology from São Paulo University to detect thrombi presence or absence. It was used hematoxylin-eosine technique, which was considered efficient. Other staining technique (Verhoff and Masom) was used when thrombi presence was observed to visualize it better.

We used the arterial blood flow analysis test (PEDERSON; SANDERS, 1999 and ACLAND, 1981) since this is a routine in surgical procedures and it is quite trustworthy.

We analyzed the presence or the absence of blood flow at 10, 20 and 30 minutes because this is the most critical period and to determine at which moment the thrombi generation might cause blood flow disturbances.

In the Group I, 10 minutes after the microanastomosis, there was lack of blood flow in five animals, which demonstrated that the vessel lesion might promote a fast thrombus formation. 20 and 30 minutes after the microanastomoses this number was 6 and 7, respectively. Animal number 2 showed thrombosis in spite of blood flow presence (PEDERSON; SANDERS, 1999 and ACLAND, 1981). The thrombus was probably in formation and, in spite of blood flow presence at 30 minutes, it should be absent rapidly. It is very difficult to make a clinical interpretation of the flow speed, but we know that a slow flow strong indicates thrombus formation. In the animal number 2 the formation of the thrombus might be considered late (after 30 minutes), and the histological exam evidenced the beginning of its formation.

In the Group II only two animals showed lack of arterial blood flow at 10 minutes. This number increased to three animals at 20 and 30 minutes. The histological exam confirmed the test performed through microsurgical tweezers (PEDERSON; SANDERS, 1999 and ACLAND, 1981).

When we compared data obtained from Group I and Group II with relationship to blood flow presence or absence at 10, 20 and 30 minutes we did not observe statistically significant differences. However, we noticed a tendency to the significance in the comparison between groups with relationship to blood flow at 30 minutes (p = 0.089). With the increased sample, we might have demonstrated the difference between Groups.

We observed a statistically significant difference with relationship to thrombi presence or absence in the histological analysis of the femoral arteries of the mice in both Groups. This means that abciximab was effective for the prevention of the thrombi formation in the femoral arteries submitted to trauma and vascular anastomosis.

We did not observe important bleeding in mice of the Group II in comparison to the Group I. However, we should consider that the surgical trauma for dissection and microanastomoses of the femoral vessels in small animals can not be enough to generate complications related with bleeding.

In this experimental model, the analysis of the efficacy of this drug evidences a new perspective in the control of the platelet aggregation for the prophylaxis of the thrombosis in vascular microanastomoses. Subsequent clinical studies will be necessary to determine the limits of this drug indication.

CONCLUSION

In this experimental model, the histological study demonstrated that the administration of the drug abciximab caused a decline in the incidence of the thrombosis in the vascular microanastomoses.

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Publication Dates

• Publication in this collection
  22 May 2007
• Date of issue
  June 2000