Femoral Artery Thrombosis Model Research Articles

Vampire Bat Salivary Plasminogen Activator Evokes Minimal Bleeding Relative to Tissue-Type Plasminogen Activator as Assessed by a Rabbit Cuticle Bleeding Time Model

Cuticle bleeding time (CBT) measurements in anesthetized rabbits were performed to assess the potential bleeding risks which may accompany the administration of tissue-type plasminogen activator (tPA) or vampire bat salivary plasminogen activator (BatPA). The dose of BatPA or tPA used in this study, 42 nmol/kg, was previously shown to be efficacious using a rabbit femoral artery thrombosis model (Gardell et al, Circulation 84:244, 1991). CBT was determined by severing the apex of the nail cuticle and monitoring the time to cessation of blood flow. CBT was minimally elevated (1.6-fold, p = NS) following bolus intravenous administration of BatPA; in contrast, bolus intravenous administration of tPA dramatically elevated CBT (6.2-fold, p < 0.05). Rabbits treated with tPA, but not BatPA, displayed profound activation of systemic plasminogen and consequent degradation of Factor VIII and fibrinogen. Elevations in CBT after the administration of tPA were reversed by the replenishment of plasma Factor VIII activity to 40% of control, but were unaffected by complete replenishment of plasma fibrinogen. The results of this study suggest that the administration of BatPA, at a dose that promotes thrombolysis, may evoke a minimal bleeding risk, relative to an equi-efficacious dose of tPA. In addition, the tPA-provoked proteolytic consumption of Factor VIII may be a key contributor to the heightened bleeding risk.

Antiplatelet and antithrombotic efficacy of DMP 728, a novel platelet GPIIb/IIIa receptor antagonist.

Currently used antiplatelet drugs, including aspirin, ticlopidine, and others, are effective against certain but not all of the many endogenous platelet activators. Because of their limited efficacy, a significant number of serious thromboembolic complications still occur, highlighting the need for a more effective therapy. Thus, we have identified a systemically active peptide analogue (DMP 728) of the arginine-glycine-aspartic acid (RGD) recognition sequence that mediates the binding of ligands such as fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptors. The goals of the present study were to determine the antiplatelet and antithrombotic efficacies of DMP 728 in various arterial thrombosis models. DMP 728 demonstrated antiplatelet efficacy in vitro in inhibiting ADP-induced human platelet aggregation (IC50, 46 +/- 2 nmol/L) and fibrinogen binding to human platelets (IC50, 2.3 +/- 0.8 nmol/L) or purified human GPIIb/IIIa receptors (IC50, 0.6 +/- 0.1 nmol/L). DMP 728 demonstrated high affinity and specificity for human platelet GPIIb/IIIa over other adhesion molecules. In anesthetized mongrel dogs, DMP 728 at 0.001 to 1.0 mg/kg IV produced dose-dependent antiplatelet effects in inhibiting ex vivo platelet aggregation induced by ADP and in prolonging template bleeding time. DMP 728 effects on bleeding time prolongation were more rapidly reversible than those on platelet aggregation inhibition. A maximal antiplatelet effect for DMP 728 was demonstrated at 0.01 mg/kg IV bolus. The antithrombotic efficacy of DMP 728 was examined in vitro and in vivo after IV administration at different doses in various models of arterial thrombosis. In the coronary artery Folts model in dogs, DMP 728 demonstrated maximal antithrombotic efficacy at 0.01 mg/kg IV bolus with an ED50 of 0.005 mg/kg IV bolus in inhibiting cyclic flow reductions. Additionally, DMP 728 demonstrated 100% prevention of primary thrombosis and rethrombosis (P < .01) after treatment with different thrombolytics, including tissue plasminogen activator and streptokinase, in an electrolytically induced femoral artery thrombosis model in dogs. Acute intravenous DMP 728 administration (0.001 to 1.0 mg/kg) has dose-dependent antiplatelet and antithrombotic effects in different arterial thrombosis models. These data suggest that DMP 728, a low-molecular-weight GPIIb/IIIa receptor antagonist, may have therapeutic potential as an effective antithrombotic agent in coronary and peripheral artery thromboembolic disorders.

Effect of Dietary Docosahexaenoic Acid Supplementation on Platelet Function: Studies in the Rat Femoral Artery Thrombosis Model

We have developed a model whereby the femoral artery in an experimental animal can be occluded by a photochemical reaction between rose bengal and green light which causes endothelial injury followed by platelet adhesion, aggregation and formation of a platelet rich thrombus at the site of the reaction. Using this model, we investigated the effect of dietary docosahexaenoic acid (DHA) supplementation on platelet aggregation and on serum cholesterol and lipids. Male Wistar rats (iweeks-old) received dietary DHA supplementation (300 mg/kg/d) for 8 weeks. This regimen produced a significant (p < 0.05) decrease in serum free-cholesterol and phospholipids levels, inhibited platelet aggregation ex vivo induced by collagen in whole blood (p < 0.05) but not in platelet rich plasma, reduced thromboxane B(2) formation (p<0.01) induced by collagen in washed platelets and prolonged the time for thrombotic arterial occlusion (p<0.01) as compared with values obtained in animals on standard diet. In conclusion, dietary DHA produces antithrombotic effects such as reduction in platelet aggregability and lowering of plasma cholesterol. Whole blood where red and white blood cells can exert their influences on platelet function is a more sensitive and physiological medium than platelet rich plasma for studying the effects of antithrombotic treatments on platelet aggregability ex vivo.

Enhancement of recombinant tissue plasminogen activator-induced reperfusion by recombinant tick anticoagulant peptide, a selective factor Xa inhibitor, in a canine model of femoral arterial thrombosis

Tick anticoagulant peptide (TAP) is a 60 amino acid protein originally isolated from the soft tick, Ornithodoros moubata, which exhibits potent anticoagulant properties due to its selective inhibition of blood coagulation factor Xa. We evaluated a recombinant version of TAP (rTAP) for its ability to accelerate recombinant tissue plasminogen activator (rt-PA) -mediated lysis of an occlusive thrombus and prevent acute reocclusion in a canine model of femoral artery thrombosis. An occlusive thrombus was formed by insertion of a thrombogenic copper coil into the femoral artery of anesthetized dogs. Blood flow velocity was monitored directly and continuously by Doppler flowmetry. 60 min after occlusion, dogs received an i.v. infusion of either saline or rTAP (0.5, 2.5 or 8.0 μg/kg/min), followed 45 min later by rt-PA (0.8 mg/kg, i.v. over 90 min; n=8/group). The saline and rTAP infusions were discontinued 1 h after stopping the rt-PA. All dogs achieved reperfusion, with a time to reperfusion in the saline-treated (vehicle) group (administered rt-PA alone) of 61 ±7 min. The time to reperfusion was slightly decreased in the 0.5ug/kg/min rTAP group (47±4min, p=NS). In the groups administered rTAP at 2.5 and 8.0 ug/kg/min, significant reductions in the time to reperfusion were observed (28±4 and 32±5mins, respectively, p<0.05). Following termination of the rt-PA, all vehicle dogs reoccluded in 37±11 min. The lowest dose of rTAP, 0.5 ug/kg/min, had no effect on either the reocclusion incidence or time (8/8 in 39±7min). In contrast, the two higher doses of rTAP maintained vessel patency in all dogs during the rTAP infusion period and dramatically delayed the time to reocclusion. However, 7/8 dogs eventually reoccluded in 117±12 and 140±9min for the groups receiving rTAP infusions of 2.5 and 8.0 ug/kg/min, respectively. Maximal elevations in activated partial thromboplastin time or template bleeding time associated with the rTAP administration were only 1.3- and 1.1-fold of baseline values, respectively. The dramatic effect of factor Xa inhibition on the efficacy of rt-PA-mediated reperfusion and acute reocclusion following rt-PA suggests that factor Xa inhibition may represent a potentially useful therapeutic adjunct to thrombolytic therapy.

P-94 - Antithrombotic action of a new 1,5-benzothiazepine derivative TA-993 in a canine model of femoral arterial thrombosis.

P-94 - Antithrombotic action of a new 1,5-benzothiazepine derivative TA-993 in a canine model of femoral arterial thrombosis.

Recombinant Pro-Urokinase Requires Heparin for Optimal Clot Lysis and Restoration of Blood Flow in a Canine Femoral Artery Thrombosis Model

Heparin is often used as an adjunct to thrombolytic therapy in order to prevent reocclusion of the patent vessels in patients with thrombotic disease. Controversy exists as to whether heparin is required for effective clot lysis with tissue-type plasminogen activator, while in vitro data and small scale clinical trials have suggested an enhancement of pro-urokinase efficacy by heparin. The present study was conducted to determine whether heparin pre-treatment is required to produce optimal clot lysis and blood flow restoration in response to recombinant pro-urokinase (r-proUK). In four groups of dogs, blood clots labelled with 125Iodine were formed in the femoral artery and were monitored continuously for loss of counts as an indicator of clot lysis. Femoral artery blood flow was measured simultaneously. Group 1 received vehicle (n = 5), while group 2 was given vehicle + heparin (n = 6; 500 U bolus + 350 U/h). This dose of heparin increased the activated partial thromboplastin time (APTT) by at least 1.5 times the control level for the 4 h observation period. Group 3 received r-proUK alone at a dose of 100,000 U/kg (50% given as a 1-min bolus injection, 50% as a 30 min infusion) (n = 8), while group 4 was treated with the same dose of r-proUK in the presence of heparin as described (n = 8).2

Heparin and the thrombin inhibitor argatroban enhance fibrinolysis by infused or bolus-injected saruplase (r-scu-PA) in rabbit femoral artery thrombosis

Enhancement by anticoagulation of thrombolysis with infused or bolus-injected saruplase (r-scu-PA) has been studied using heparin and the thrombin inhibitor argatroban. In a rabbit femoral artery thrombosis model infusion of saruplase (3 – 12 mg/kg, 60 min) caused a dose-dependent thrombolysis. Reperfusion rate after infusion of 3 mg/kg saruplase alone was 3 6 , reperfusion time 42 ± 3 min and reocclusion rate 2 3 ; final patency rate at 120 min was 17 %. Combination of 3 mg/kg saruplase with heparin (150 U/kg + 100 U/kg hr i.v.; 5.3-fold PTT-prolongation) resulted in a reperfusion rate of 6 6 after a reperfusion time of 39 ± 7 min; reocclusion rate was 3 6 and final patency rate was 50 %. Argatroban (1 mg/kg + 3 mg/kg.hr i.v.; 2.3-fold PTT prolongation) in combination with saruplase resulted in a reperfusion rate of 6 6 after 26 ± 5 min; no reocclusion occured and final patency rate was 100 % (p < 0.05 vs saruplase alone). Bolus injection of 6 mg/kg saruplase achieved reperfusion in 5 6 arteries after 15 ± 3 min, but reocclusion rate was 4 5 ; final patency rate was 17 %. Combination of bolus-injected saruplase with heparin resulted in a reperfusion rate of 4 6 after 8 ± 3 min and no reocclusion occured; patency rate was 67 %. With combination of argatroban and bolus-injected saruplase 6 6 arteries were reperfused after 8 ± 3 min; reocclusion was prevented and final patency rate was 100 % (p < 0.05 vs saruplase-bolus alone). Systemic fibrinogenolysis was more pronounced with bolus injection than infusion of saruplase. The results indicate that arterial thrombolysis with saruplase can be enhanced by heparin and the thrombin inhibitor argatroban. The bolus injection of saruplase resulted in persistent reperfusion when simultaneous anticoagulation was performed. Despite less PTT prolongation, enhancement of saruplase-induced thrombolysis was more effective with argatroban than with heparin in rabbit femoral artery thrombosis.

Effective thrombolysis without marked plasminemia after bolus intravenous administration of vampire bat salivary plasminogen activator in rabbits.

The use of recombinant tissue-type plasminogen activator (t-PA) in thrombolytic therapy is frequently associated with significant fibrinogenolysis. In contrast, recombinant vampire bat salivary plasminogen activator (Bat-PA) displays strict fibrin specificity, an attribute that could be desirable in a fibrinolytic agent. The efficacy and fibrin selectivity of Bat-PA was evaluated and compared with that of t-PA using a rabbit model of femoral arterial thrombosis. Administration of 8.1, 14, and 42 nmol Bat-PA/kg by bolus intravenous injection restored flow in 50%, 75%, and 80% of the rabbits, respectively. The incidence of reperfusion after bolus intravenous injection of 14 and 42 nmol t-PA/kg was 15% and 78%, respectively. The maximal femoral artery reperfusion flows were equivalent after treatment with 42 nmol Bat-PA/kg or 42 nmol t-PA/kg, but the time to reach maximal flow for Bat-PA was approximately one half that of t-PA. Furthermore, the rapid restoration of flow by 42 nmol Bat-PA/kg, in contrast to equimolar t-PA, was accomplished without fibrinogenolysis and with only small decreases in the plasminogen and alpha 2-antiplasmin levels. Equipotent doses of Bat-PA and t-PA both resulted in approximate 2.5-fold increases in the template bleeding times of aspirin-pretreated rabbits. The clearance of Bat-PA from rabbits exhibited biexponential elimination kinetics; approximately 80% was cleared by the relatively slow beta phase (half-life of 17.1 minutes). Overall, Bat-PA was cleared approximately fourfold slower than t-PA. Bolus intravenous administration of Bat-PA would facilitate prompt initiation of thrombolytic therapy, and the avoidance of plasminemia could result in fewer and less severe bleeding complications.

Enhanced thrombolysis by a factor XIIIa inhibitor in a rabbit model of femoral artery thrombosis

Factor XIIIa (FXIII a) catalyzes covalent crosslinking reactions of fibrin, affording the clot additional structural stability and resistance to plasmin-mediated degradation. Thus, inhibition of FXIIIa may render thrombi more susceptible to tissue-plasminogen activator (t-PA)-induced thrombolysis in vivo . We therefore examined thrombus weight and time to lysis in anesthetized rabbits undergoing arterial thrombosis produced by insertion of a copper coil into the lumen of the right femoral artery. The effects of t-PA alone (started 30 min after coil insertion) or in combination with a FXIIIa inhibitor (L722151) started 15 min before, 8 min after or 20 min after coil insertion were determined. Although t-PA alone (2 mg/kg over 2 hrs) lowered thrombus weight significantly, there was no evidence of flow restoration. Addition of L722151 to t-PA before, or 8 min after coil insertion, further lowered thrombus weights and produced thrombolysis in 50% of the animals. This beneficial effect was lost when L722151 administration was delayed until 20 min after thrombus formation, suggesting that the type(s) of crosslinking inhibited by L722151 was complete by this time. Infusion of L722151 alone had no significant effect on thrombus weight. These results demonstrate a time-dependent facilitation of t-PA-induced arterial thrombolysis by FXIII a inhibition in a small animal model.

Prevention of Reocclusion by MCI-9038, a Thrombin Inhibitor, Following t-PA-lnduced Thrombolysis in a Canine Model of Femoral Arterial Thrombosis

We compared a selective thrombin inhibitor (MCI-9038; Argatroban), a thromboxane A2 (TXA2) receptor antagonist (L-670,596) and a serotonin-2 receptor antagonist (ketanserin) for their ability to hasten clot lysis and delay reocclusion in a canine model of femoral arterial thrombosis. Occlusive thrombosis was induced by insertion of a thrombogenic copper coil. Femoral arterial blood flow velocity (FABFV) was monitored directly and continuously by Doppler flowmetry. Thrombolysis was induced with tissue plasminogen activator (t-PA; 0.8 mg/kg, i.v.), starting 60 min after thrombotic occlusion and continued for 90 min. Ten minutes after occlusion, dogs received an intravenous infusion of either vehicle, MCI-9038 (10 micrograms kg-1 min-1), ketanserin (0.1 mg/kg bolus plus 5 micrograms kg-1 min-1), L-670,596 (1 mg/kg bolus plus 17 micrograms kg-1 min-1) or a combination of L-670,596 and ketanserin. All infusions were discontinued 1 h after stopping the t-PA, and were followed by a 30 min observation period. The times to thrombolysis were similar for all treatments (mean +/- SEM = 47 +/- 3; all groups). MCI-9038 prevented reocclusion, defined as permanent cessation of FABFV during the hour after stopping the t-PA. All dogs receiving MCI-9038 reoccluded within 30 min after stopping its infusion (71 +/- 3 min). Reocclusion occurred in all other dogs, except one vehicle-treated dog and a second dog that received L-670,596 plus ketanserin. Vehicle-treated dogs reoccluded within 23 +/- 8 min. Reocclusion was not delayed significantly by ketanserin, L-670,596 or the combination of the two.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined mechanical and chemical thrombolysis in an experimental animal model: Evaluation by angiography and angioscopy

In an experimental animal model of femoral artery thrombosis, contrast angiography was compared to intravascular angioscopy. Additionally, the effect of mechanical, rotational thrombectomy and the additive benefit of the administration of intravascular streptokinase were assessed by means of both procedures. After external forceps crush injury alone, contrast angiograms were generally normal (6 of 14) or showed minimal luminal irregularity (3 of 14), and 5 of 14 had 30% to 50% stenosis. With angioscopy, none appeared normal, and 14 of 14 showed thrombi layered along the wall, as well as intimal flaps, and 6 of 14 had partially occlusive thrombi (p less than 0.001 angiography vs angioscopy). After 2-hour occlusion and injection of thrombin into the injured segment, angiographic total (5 of 14), subtotal (3 of 14), or partial thrombotic occlusions (5 of 14) were created. Angioscopy showed similar results, except that total occlusions were classed as subtotal occlusions. After rotational thrombectomy, most arteries again appeared normal by contrast angiography (6 of 11) but none were angioscopically normal (p less than 0.006). Streptokinase, administered after rotational thrombectomy in seven arteries, normalized one 30% angiographic stenosis; there were no other angiographic changes. Findings with angioscopy were also unchanged. We conclude that in the diagnosis and treatment of intravascular thrombosis, angioscopy is generally more sensitive in the detection of intravascular thrombi, with the exception of total thrombotic occlusions. Angioscopy was uniquely effective in identifying subintimal flaps, which were never identified by angiography. In this model, streptokinase provided little or no additional thrombolytic benefit to mechanical thrombectomy alone.

EFFECT OF SELECTIVE ENDOPEROXIDE/THROMBOXANE A2 RECEPTOR ANTAGONISM WITH SULOTROBAN ON tPA‐INDUCED THROMBOLYSIS IN A RABBIT MODEL OF FEMORAL ARTERIAL THROMBOSIS

The thrombolytic efficacy of recombinant tissue-type plasminogen activator (tPA) in the presence and absence of the selective endoperoxide/thromboxane A2 (TXA2) receptor antagonist, sulotroban (BM 13.177, SK&F 95587) was studied in a model of femoral artery thrombosis in the anesthetized rabbit. The thrombus was formed by injection of thrombin, CaCl2 and whole blood into an isolated segment of the femoral artery. After 30 min of stable thrombotic occlusion of the femoral artery, tPA was infused IV for 90 min at doses of 5.0, 7.5 and 10.0 micrograms/kg/min. In other experiments, sulotroban was administered as a bolus dose of 1 mg/kg/IV, followed by a constant infusion of 1 mg/kg/hr concurrent with tPA infusion. Sulotroban had no effect on the incidence of tPA-induced reperfusion at any dose studied or on residual clot weight. However, at a tPA dose of 10 micrograms/kg/min, IV lysis time was reduced in sulotroban treated animals from 65 min to 29 min (P less than 0.05), and the magnitude of femoral artery blood flow achieved as a result of tPA-induced reperfusion was greater in sulotroban-treated animals. These data suggest that adjunctive therapy with a selective endoperoxide/TXA2 antagonist improves the response to tPA when tPA is administered at a maximal or near maximally effective pharmacological dose.

The Usefulness of Systemic Administration of Recombinant Human Tissue-Type Plasminogen Activator in Femoral Arterial Thrombolysis of Rabbits

The thrombolytic effect of locally or systemically administered recombinant human tissue-type plasminogen activator (rt-PA) was investigated in comparison with the effect of tissue culture urokinase (TCUK), using a model of femoral artery thrombosis in rabbits. An 125l-labeled fibrinogen thrombus was formed in the femoral artery following injury of the intima by diluted sulfuric acid, and thrombolytic activity was evaluated one hour after the end of infusion of the agents. Local infusion of rt-PA (500-10,000 lU/kg) and TCUK (10.000 lU/kg) induced a marked thrombolysis. When rt-PA and TCUK were injected systemically in a high dose (200,000 lU/kg), rt-PA but not TCUK had a significant thrombolytic activity. In these cases, rt-PA was not accompanied by systemic activation of the fibrinolytic system, as evaluated by unaltered levels of α2-antiplasmin in the plasma, while TCUK led to a substantial decrease in the α2antiplasmin level. These results suggest that systemically administered rt-PA but not TCUK induce a significant thrombolysis without systemic activation of the fibrinolytic system in cases of peripheral artery thrombosis.

The usefulness of systemic administration of recombinant human tissue-type plasminogen activator in femoral arterial thrombolysis of rabbits.

The thrombolytic effect of locally or systemically administered recombinant human tissue-type plasminogen activator (rt-PA) was investigated in comparison with the effect of tissue culture urokinase (TCUK), using a model of femoral artery thrombosis in rabbits. An 125I-labeled fibrinogen thrombus was formed in the femoral artery following injury of the intima by diluted sulfuric acid, and thrombolytic activity was evaluated one hour after the end of infusion of the agents. Local infusion of rt-PA (500-10,000 IU/kg) and TCUK (10,000 IU/kg) induced a marked thrombolysis. When rt-PA and TCUK were injected systemically in a high dose (200,000 IU/kg), rt-PA but not TCUK had a significant thrombolytic activity. In these cases, rt-PA was not accompanied by systemic activation of the fibrinolytic system, as evaluated by unaltered levels of alpha 2-antiplasmin in the plasma, while TCUK led to a substantial decrease in the alpha 2-antiplasmin level. These results suggest that systemically administered rt-PA but not TCUK induce a significant thrombolysis without systemic activation of the fibrinolytic system in cases of peripheral artery thrombosis.

Effect of thromboxane synthase inhibition on the thrombolytic action of tissue-type plasminogen activator in a rabbit model of peripheral arterial thrombosis

The thrombolytic efficacy of recombinant tissue-type plasminogen activator (tPA) in the presence and absence of a thromboxane synthase inhibitor was studied in a model of femoral artery thrombosis in the anesthetized rabbit. The thrombus was formed by injection of thrombin and whole blood into an isolated segment of the femoral artery. After 30 min of stable thrombotic occlusion of the femoral artery, sodium heparin (300 U/kg, i.v.) was administered and tPA was infused locally to the site of the thrombus for 30 min at 0.01, 0.10 or 1.0 μg/kg/min. In other experiments, CGS 13080, a selective thromboxane synthase inhibitor, was administered at a dose of 2 mg/kg i.v., 5 min before tPA was infused and at the end of the 30 min tPA infusion. Pretreatment with CGS 13080 resulted in a shorter time to tPA-induced reperfusion, greater incidence of reperfusion and increased the magnitude of femoral artery blood flow achieved after effective thrombolysis. Furthermore, pretreatment with CGS 13080 resulted in a greater than 10-fold enhancement in the effective dose of tPA. These data indicate that thromboxane synthase inhibition may be beneficial as an adjunct to thrombolytic therapy with tPA.

ヒトＭｅｌａｎｏｍａ細胞株由来Ｔｉｓｓｕｅ‐ｔｙｐｅ Ｐｌａｓｍｉｎｏｇｅｎ Ａｃｔｉｖａｔｏｒ（ｔ‐ＰＡ）のウサギ動静脈血栓に及ぼす影響

We have examined the thrombolytic effect of 1hr intravenous infusion of human tissue-type plasminogen activator (t-PA) and purified two chain urokinase (u-PA) on rabbit thrombosis models in juglar vein and femoral artery. In the juglar vein thrombosis model t-PA of 100×1003IU/kg showed 51% thrombolysis without severe systemic fibrinolysis. On the other hand, u-PA of 500×103IU/kg elicited 69% thrombolysis with significant and dose dependent reduction of α2-plasmin inhibitor, plasminogen and fibrinogen levels. In the femoral artery thrombosis model the similar results were observed. Accordingly, it can be concluded that t-PA is more specific and effective thrombolytic agent than u-PA in rabbit models.