Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

A functional model of microvascular thrombosis

A new model of microvascular thrombosis is presented, with the evaluation of single-dose heparin in the prevention of microvascular thrombosis. The technique, which involves arterial crushing and an arteriotomy with intimal abrasion, was performed on the superficial femoral artery of the rat. The model was applied to a series of 30 consecutive rat superficial femoral arteries. A 100 percent thrombosis rate was seen immediately and at 24 hours in 10 nonheparinized animals. An operator control group of 10 vessels without intimal abrasion had a patency rate of 100 percent immediately and at 24 hours. Ten vessels following single-dose heparin and intimal abrasion were all patent initially, with 7 remaining patent at 24 hours. Reproducibility of the model was documented by a second operator with similar results. Utilizing this model, single-dose heparin was effective in maintaining vessel patency.     

Experimental microvascular polytetrafluoroethylene grafts: 6-month patency

The efficacy of 1-mm internal-diameter polytetrafluoroethylene as a microvascular prosthesis is unclear. In this study, 8-mm-long segments of this material were implanted into the femoral arteries of 30 rats. Animals were examined every 2 weeks up to 6 months by Doppler ultrasound. Cumulative patency by the life-table method was 86.7 percent at 6 months. There were 26 patent grafts, 2 occlusions, and 2 deaths. Intimal hyperplasia adjacent to the anastomoses was seen in the native arteries. The pseudointima lining the grafts was cellular near the anastomoses but usually acellular in midgraft regions. It is concluded that if early failure does not occur, then good long-term patency is possible with 1-mm polytetrafluoroethylene in this setting and that patency is not dependent on development of a cellular pseudointima. Longer graft segments should be evaluated in future studies.     

Therapeutic value of intravenous heparin in microvascular surgery: an experimental vascular thrombosis study

In an attempt to decrease a 10 to 15 percent vascular thrombosis rate leading to graft occlusion, low-dose human-grade heparin was studied to determine if carefully monitored intravenous therapy would increase 7-day patency in a known potent thrombosis model. In New Zealand white rabbits, the type of infusate administered intravenously, either saline (30 animals) or heparin (35 animals), was selected at random after completing a 2-mm arterial inversion graft in the femoral artery. A 72-hour infusion was used in all animals; the control group received sterile saline and the experimental group received a heparin infusion at 45 microliters per hour after a 500-unit bolus. All grafts in both groups were patent at the time of groin closure. Patency in the heparin-perfused group was 67 percent (24 of 35) as compared to 19 percent (6 of 30) in the control group (p less than 0.05) 1 week postoperatively. Scanning electron microscopy showed significantly less dense fibrin deposition and a decrease in the number of aggregated platelets in the heparin-perfused grafts. Partial tissue thromboplastin time values in the experimental group ranged between 55 and 75 seconds (control 20 to 25 seconds). We have shown that heparin, an inexpensive and readily available agent, maintains 1-week microarterial patency and results in few complications in a reliable, reproducible, and versatile thrombosis model. The clinical ramifications of using an antiplatelet agent that diminishes fibrin deposition in microsurgery are apparent.     

Experimental effects of heparin or magnesium sulfate on the patency of microvascular anastomoses.

Partial amputations of the hind legs were done in 44 rats. The femoral arteries and veins were repaired by microvascular techniques. The systemic use of heparin, the local use of magnesium sulfate, and the combined use of the two drugs, were evaluated as to their influence on preventing thrombosis in these microvascular anastomoses. No benefit could be demonstrated from the use of these drugs.     

"No suture" microanastomosis using Vicryl rings and fibrin adhesive system: an unsuccessful attempt

Vascular repairs using a combination of Vicryl rings to evert vessel edges and the Tisseel two-component fibrin adhesive system failed to achieve patent microanastomosis without using stay sutures. The bonding force of Tisseel alone was not sufficient to hold the cut ends of rat femoral arteries and veins together. All 10 arterial and 8 venous end-to-end anastomoses separated under normal intraluminal pressure immediately after the removal of microclamps. This method was found to be an unacceptable alternative to the conventional suturing method.     

The effect of established infection on microvascular surgery

The success of microvascular anastomoses in the presence of staphylococcal infection was studied using rat femoral arteries. There was a spontaneous thrombosis rate of 19 percent in normal vessels that traversed the area of infection. Vessels with an anastomosis outside the area of infection had a similar thrombosis rate, but if the anastomotic site was within the infected area itself, the thrombosis rate increased to 75 percent. Inflammatory changes with subsequent fibrosis in the media and adventitia appeared responsible for the thrombosis. The intima was unaffected by the presence of infection. This study suggests that when a microvascular anastomosis is necessary in the presence of infection, the anastomosis should be placed outside the area of infection with a pedicle to traverse the infected area.     

Local subcutaneous heparin as treatment for venous insufficiency in replanted digits.

In the treatment of venous insufficiency unsuitable for surgical correction in replanted digits, a small ungual window was surgically created to infiltrate subcutaneous heparin in the congested digit. The initial heparin dose was 1000 units. This dose made possible a continuous bleeding during 24 to 48 hours, solely through the ungual window. Further doses were applied based on the degree of congestion of the replanted digit, but usually it was necessary to infiltrate up to 500 units of heparin every 24 to 48 hours until vascular stability was achieved. Three patients were treated with this technique. One opted for quitting the treatment. A replanted thumb suffered venous congestion on the seventh postoperative day and was treated with local subcutaneous heparin for 3 days. A replanted fingertip suffered venous thrombosis 24 hours after surgery and was treated likewise for 18 days. In these two patients, success was attained. Blood transfusions were carried out in the latter two, and none had any systemic changes in partial thromboplastin or thrombin time. This treatment is based on the mechanism of action of heparin at high doses but applied only to the congested segment. Besides their anticoagulant effect through antithrombin, high doses of heparin slow platelet aggregation, may induce angiogenesis, and have a longer-than-normal half-life. With the above technique, heparin has been applied to the congested segment at an approximate dose of 33,000 to 40,000 units/kg, and continuous bleeding solely through the ungual window for 24 to 48 hours has been achieved, which has allowed us to save two replanted segments with no complications at all. This method may offer another alternative for the medical treatment of venous insufficiency in replanted segments.     

Anastomosis of vessels of unequal diameter using an interpositional vein graft

In this study, 100 rabbits were used to assess the efficacy of five different methods of microvascular anastomosis where a vessel diameter discrepancy of 5:1 existed. The inferior vena cava of the rabbit was used as a graft in the femoral artery. In 50 percent of the rabbits the graft was reversed to assess the effects on flow. When explored between 7 and 10 days after anastomosis, an overall patency rate of 96 percent was recorded. Three grafts were not patent in the reversed group and one was not patent in the nonreversed group. There was no significant statistical difference in patency rates between any of the groups, as calculated by the Fisher's exact probability test. The tapered end-to-end and side-to-end anastomoses were found to be the most rapid and simplest methods to perform.     

Prevention of microvascular thrombosis with low-dose tissue plasminogen activator.

In a blind, randomized study, two groups, each of seven rabbits, were treated with either a very low dose of human melanoma cell line-derived tissue-type plasminogen activator (t-PA) or isotonic saline. t-PA (0.067 mg/kg of body weight) was administered intraaortically, 20 percent being given as a 30-second "bolus" infusion just prior to the reperfusion of intimectomized central ear arteries and the rest as a continuous infusion during the next 2 hours. Arteriotomic bleeding times, accumulations of 32P-labeled platelets, patency, and sizes of thrombus deposits 2 hours after reperfusion were recorded. To confirm the presence of tissue plasminogen activator in plasma, fibrin-plate lysis assays of arterial plasma were performed immediately before and 1/2 hour and 2 hours after starting drug infusion. Arteriotomic bleeding times were similar in both groups. Transient "oozing" from wound edges occurred in 40 percent of rabbits treated with tissue plasminogen activator. Patency was significantly increased and thrombus deposits were smaller in the tissue plasminogen activator group. Plasma from animals treated with tissue plasminogen activator caused massive lysis of fibrin plates, whereas plasma from control animals caused little or no lysis. Platelet accumulations were very similar in both groups, indicating that occlusive thrombi mainly consisted of other elements than platelets (e.g., fibrin and red cells). Scanning electron microscopy showed normally adhering and aggregating platelets in both groups. This study shows that mild fibrinolytic stimulation with tissue plasminogen activator significantly improves patency in severely traumatized small-caliber arteries and indicates that such treatment may be one approach to prevent thrombosis at microvascular anastomotic sites.     

A phase II trial of intraluminal irrigation with recombinant human tissue factor pathway inhibitor to prevent thrombosis in free flap surgery

A multicenter, multinational, blinded, randomized, parallel-group, phase II study was conducted to investigate the use of recombinant human tissue factor pathway inhibitor (rhTFPI; SC-59735) as an antithrombotic additive to the intraluminal irrigating solution during microvascular anastomosis in free flap reconstructive surgery. A total of 622 patients undergoing free flap reconstruction were randomly assigned to three groups. For each group, a different intraluminal irrigating solution was administered at completion of the microvascular arterial and venous anastomoses and before blood flow to the flap was reestablished: rhTFPI at a concentration of 0.05 or 0.15 mg/ml (low-dose or high-dose group, respectively) or heparin at a concentration of 100 U/ml (current-standard-of-practice group). There were no other differences in treatment among the groups. Patient characteristics, risk factors, and surgical techniques used were similar among all three groups. Flap failure was lower (2 percent) in the low-dose rhTFPI group than in the high-dose rhTFPI (6 percent) and heparin (5 percent) groups, but this difference was not statistically significant (p = 0.069). There were no significant differences in the rate of intraoperative revisions of vessel anastomoses (11 percent, 12 percent, and 13 percent) or postoperative thrombosis (8 percent, 8 percent, and 7 percent) among the low-dose rhTFPI, high-dose rhTFPI, and heparin groups, respectively. The rate of postoperative wound hematoma was significantly lower in the low-dose rhTFPI group (3 percent) than in the high-dose rhTFPI (8 percent) and heparin (9 percent) groups (p = 0.040). There were no differences in blood chemistry or coagulation values among the three study groups. Other than hematomas, there were no differences in the incidence or severity of adverse reactions among the three groups. It is concluded that use of rhTFPI as an intraluminal irrigant during free flap reconstruction is safe, well tolerated, and as efficacious as use of heparin for preventing thrombotic complications during and after the operation. Furthermore, the lower dose of rhTFPI (0.05 mg/ml) may reduce the occurrence of postoperative hematoma and help prevent flap failure.     

A new technique for microvascular anastomosis: external metallic circle

Vessel anastomosis is the most critical step in free tissue transfers and replantation surgery. We report on a new microvascular anastomosis technique that uses a metallic circle around the anastomotic circumference. Sutures are first passed inside the circle and tied outside and over the circle so as to stretch open the anastomotic site. By retraction of vessel ends, the circle is totally exteriorized and thus there is no contact with blood. In 48 rats, the external circle method was compared with the conventional technique for constructing end-to-end anastomosis between carotid arteries (1 to 1.2 mm) and femoral veins (1 to 1.5 mm). The external circle method proved to be superior to the conventional end-to-end technique in speed of execution for both arterial and venous anastomoses. Patency rates at the third week were significantly higher in the venous group using the metallic circle (100 percent versus 70.8 percent, p < 0.05). This new method may be applicable in clinical microvascular surgery.     

End-to-end versus end-to-side microvascular anastomosis patency in experimental venous repairs

In this experimental study, venous end-to-end and end-to-side microvascular anastomoses in similar and diameter-discrepant vessels were compared. In 50 rats, end-to-end microvascular repair of the divided epigastric vein and end-to-side repair of the epigastric vein into the femoral vein showed 5-day patency rates of 75 and 88 percent, respectively. These data are not statistically different. In 20 rats, microvascular repair of end epigastric to end femoral veins (size discrepant) and end epigastric to side femoral veins showed 5-day patency rates of 50 and 85 percent, respectively. These data are statistically different (p less than 0.05). We conclude from these experimental data that end-to-side venous repairs may be useful in lowering the anastomosis thrombosis rate seen when size-discrepant veins are repaired.     

Comparison of the thrombogenicity of internationally available fibrin sealants in an established microsurgical model.

Previous studies comparing the thrombotic complications of cryoprecipitated fibrin sealant containing bovine thrombin on microvascular venous anastomoses in a rat epigastric free flap model revealed deleterious outcomes regarding flap survival with higher concentrations of topical bovine thrombin. This study was designed to compare three internationally available fibrin sealants, one experimental fibrin monomer sealant that does not require thrombin, and human thrombin alone as to their effects on the survival of an established rat epigastric free flap model. Ninety Sprague-Dawley rats (400 to 600 g) were prepared for abdominal surgery, and an epigastric-based skin flap was raised. The single vein draining the flap was clamped, divided, and reconnected using standard microvascular suturing techniques. Before release of the clamps, the chosen additive was applied precisely to the anastomosis. Additional material was then added to the raw surface of the flap. The animals were divided into seven treatment groups, each receiving 1 ml of commercial or investigational fibrin sealant or human thrombin alone: one control group receiving no additive treatment, four fibrin sealant groups receiving treatment with commercial or investigational fibrin sealant preparations, and two groups receiving different concentrations (500 IU/ml and 1000 IU/ml) of human thrombin applied to the anastomoses and the surrounding tissue. Flap survival was assessed at 7 days postoperatively. This study supports the contention that microvascular free flap survival based on microvascular venous anastomotic patency was adversely effected by high concentrations of thrombin. Lower concentrations (500 IU/ml and less) of thrombin did not seem to affect flap survival. One test product was composed of a fibrin monomer sealant, which obviates the need for the thrombin additive. This group's survival rate was not statistically different from that of the control group. Thus, for microvascular anastomoses, lower concentrations of thrombin or a sealant devoid of thrombin seem to be best for microvascular anastomotic patency.     

Thrombotic thrombocytopenic purpura: a syndrome of intravascular platelet consumption.

In four of five patients with thrombotic thrombocytopenic purpura (TTP) in whom serial tests of hemostatic function were performed, severe thrombocytopenia, normal plasma fibrinogen concentrations and mildly increased concentrations of fibrinogen/fibrin degradation products were observed. Widespread platelet thrombi were found in arterioles and capillaries. Fibrin could be seen around some of the platelet clumps and was the main component in a small number of the thrombi in two patients. The observations show that TTP is a disorder in which intravascular platelet consumption results in disseminated platelet thrombosis. The coagulation system is apparently activated secondarily to platelet aggregation and variable quantities of fibrin are incorporated into the thrombi. Clinical improvement resulted from combined therapy with corticosteroids, heparin and drugs that suppress platelet function.     

Mechanical anastomosis of small arteries and veins with the unilink apparatus: a histologic and scanning electron microscopic study

A new method for mechanical anastomosis of small vessels--the Unilink device--has been tested in 23 rabbits. A total of 81 arterial and venous anastomoses were performed. One of the arterial anastomoses were thrombotized, while the remaining 80 anastomoses were fully patent at 2 or 16 weeks. The repair process at the anastomotic site was very rapid both in arteries and veins. The endothelialization was complete at 2 weeks, but a marked atrophy of the media was noted in the arterial anastomoses. The same phenomenon was observed in the venous anastomosis, but to a much lesser degree. Thrombus formation was extremely rare, and the atrophy of the media did not seem to affect the patency rate. The experiment has confirmed that the Unilink method provides a very safe, fast, and simple way to perform microvascular anastomoses.     

Potentiation of phosphodiesterase inhibitor antithrombotic activity with alpha-2 adrenergic blockade.

The antithrombotic activity of pelrinone, a phosphodiesterase III inhibitor was examined in a canine model of coronary thrombosis that uses electrical current to injure the coronary endothelium. Ninety percent of vehicle treated animals developed complete coronary occlusion and thrombus mass was 32.0 +/- 5.8 mg. In a group of animals treated with zomepirac, 10 mg/kg i.v., included as a positive control, thrombus mass was decreased to 10.3 +/- 3.3 mg and incidence of occlusion was reduced to 37.5%. Pelrinone, 5.0 mg/kg i.v. decreased the incidence of occlusion to 50%, thrombus mass to 21.3 +/- 8.3 mg and inhibited platelet aggregation to collagen, ADP and arachidonic acid by 80%, 54% and 87% of baseline, respectively. When yohimbine, an alpha 2-adrenergic antagonist, was co-administered (2.0 mg/kg at the beginning of the experiment +0.5 mg/kg halfway through the experiment) with the same dose of pelrinone, thrombus mass was decreased to 1.0 +/- 0.5 mg and none of the animals developed coronary occlusion. Yohimbine administration by itself at 2.0-3.0 mg/kg showed no evidence of antithrombotic activity (thrombus mass = 32.8 +/- 8.0 mg, incidence of occlusion = 100%). This dose of yohimbine inhibited significantly ADP-induced aggregation in the presence of epinephrine. These results demonstrate that, even though this dose of pelrinone elicited near maximal inhibition of platelet aggregation, the concurrent administration of an alpha 2-adrenergic antagonist was able to potentiate markedly the phosphodiesterase inhibitor antithrombotic activity.     

Anticoagulant effects of the heparin-proline complex

In the present work, the formation of the proline-heparin complex has been established. The way to synthesize the complex in vitro has been developed. The complex synthesized with the molar ratio of proline to heparin of 3: 1 extended the time of formation of a fibrin clot, reduced platelet aggregation, and showed a lytic effect towards nonstabilized fibrin in vitro conditions. Ten minutes after intravenous administration of the proline-heparin complex, there was an increase in levels of fibrin-depolymerization, anticoagulant, antifibrin-stabilizing and antiplatelet activity in blood of animals, whereas its components, proline and heparin, did not have such effects.     

Microvascular anastomoses in irradiated vessels: a comparison between the Unilink system and sutures

A new mechanical device (the Unilink system) was compared to conventional suture anastomoses in irradiated microvessels. Twenty rabbits received a single radiation dose of 20 Gy from a 7-MeV electron source through an anterior neck field. One and 6 months following irradiation, the carotid arteries and facial veins were divided and anastomosed on one side with the Unilink system and on the other side with suture technique. At sacrifice 4 weeks postoperatively, all vessels were evaluated for patency and histologic changes associated with radiation and anastomotic trauma. Histology disclosed severe radiation changes. Also, intimal hyperplasia was consistently found at the anastomotic sites in the arteries, while it was totally absent in the venous anastomoses. Occlusive thrombosis was found in two arteries, one anastomosed with the Unilink system and one sutured. Two other arteries, one from each group, had subtotal occlusions at the anastomotic site. No occlusions occurred in any of the venous anastomoses. The overall patency in this study was 97.5 percent, with no difference between the two techniques.     

Eversion with four sutures: an easy, fast, and reliable technique for microvascular anastomosis

In this study, a microvascular anastomosing technique called "eversion with four sutures" is introduced. For microvascular anastomosis, this technique requires fishmouth incisions at both vessel ends and the completion of four sutures. In 120 Wistar-Albino rats, 120 eversion and 120 conventional anastomoses were done in 240 femoral arteries. Each rat received both treatments. Operating time, bleeding time, number of sutures used, patency rates, and pseudoaneurysm formation were analyzed statistically; healing was evaluated with both light and electron microscopy. When compared with the conventional technique using nine sutures, the eversion with four sutures technique was found to be a faster and easier method of anastomosis and as reliable as the conventional technique. Without compromising patency rates, bleeding time, or rates of pseudoaneurysm formation, anastomosis time and amount of suture material exposed to the lumen were significantly reduced when using this technique. In conclusion, the authors think that eversion with four sutures is a reliable alternative to the conventional suturing technique, especially for emergency cases that require multiple microvascular anastomoses.