Heparin cofactor II inhibits thrombus formation in a rat thrombosis model

Heparin cofactor II is postulated to be an extravascular thrombin inhibitor that is physiologically stimulated by dermatan sulfate. However, the role of heparin cofactor II has not yet been clearly demonstrated in vivo. In this study, we estimated the antithrombotic effect of heparin cofactor II administered exogenously in a rat model of thrombosis. Thrombus was induced in the rat femoral artery by endothelial damage due to the photochemical reaction between systemically injected rose bengal and transillumination with green light. Pretreatment with heparin cofactor II significantly prolonged the time required to occlude the femoral artery (occlusion time) in a dose-dependent manner. At an effective dose in this thrombosis model, heparin cofactor II did not prolong the activated partial thromboplastin time and the prothrombin time in normal rats. Argatroban, a selective synthetic thrombin inhibitor, significantly prolonged the occlusion time. However, argatroban also prolonged the activated partial thromboplastin time and prothrombin time at an effective dose. These results suggest that the administration of heparin cofactor II in vivo effectively inhibited thrombus formation on the vessel walls whose endothelium is damaged without a prolongation of the coagulation time while heparin cofactor II may also inhibit the thrombin activity in the subendothelial tissue in vivo.     

Comparison of the inhibitory effects of the TXA2 receptor antagonist, vapiprost, and other antiplatelet drugs on arterial thrombosis in rats: possible role of TXA2.

The antithrombotic effect of the thromboxane A2 receptor antagonist, vapiprost, was compared with those of other antiplatelet drugs using an arterial thrombosis model which utilized photochemical reaction in the rat femoral artery. Vapiprost prolonged the time required to occlude the artery with thrombus and inhibited collagen-induced rat platelet aggregation in whole blood ex vivo, in a dose-dependent manner. The potency ranking of antithrombotic effect was vapiprost > ketanserin (serotonin 5-HT2 receptor antagonist) > ticlopidine (inhibitor of ADP-induced platelet aggregation) = dipyridamole (adenosine uptake inhibitor) > aspirin (cyclooxygenase inhibitor). On the other hand, the ranking of antiplatelet effect was ticlopidine > or = vapiprost > or = aspirin. Ketanserin and dipyridamole were ineffective. Relative to their antiplatelet effect, vapiprost and ketanserin had powerful antithrombotic effects. It is possible that the potent antithrombotic effects of vapiprost and ketanserin in vivo reflect the ability of these drugs to inhibit mediator-induced vascular contractions in addition to platelet aggregation. The results of the present study also suggest that TXA2 may play an important role in thrombogenesis in rats.     

Thrombin inhibition compared with other antithrombotic drugs in rats.

An aspirin-sensitive model of arterial thrombosis suitable for rapid evaluation of antithrombotic drugs was developed and characterized in anesthetized rats. Carotid artery thrombi were formed in response to electrical stimulation and were occlusive in 84% of vehicle-treated rats. Light and electron microscopy revealed these thrombi to be platelet-rich and fibrin-rich masses adherent to the injured vessel wall. Intravenous administration of aspirin (10 mg/kg), heparin (300 U/kg), a thromboxane (Tx) A2-receptor antagonist (SQ 29,548, 0.2 mg/kg + 0.2 mg/kg/hr), or the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK, 52 micrograms/kg/min) decreased average thrombus weight by 35, 50, 57 and 94%, respectively. Each of these drugs also reduced the frequency of occlusion to < 25%. In contrast, thrombus weight and vessel occlusion were not decreased by a serotonin antagonist (ketanserin, 0.3 mg/kg, i.v.), or after 14 days of oral dosing with either the calcium antagonist diltiazem (60 mg/kg) or SQ 33,351 (30 mg/kg).     

An improved photochemical model of embolic cerebral infarction in rats.

To provide further evidence that the multiple cerebral infarcts found in rats following photochemical damage to the carotid artery are caused by emboli and to eliminate the systemic hypotension and heating of the blood reported with the previous photochemical embolic stroke model (rose bengal and a green laser), I have modified the photochemical technique. Brain pathology was studied in 18 Wistar rats following carotid artery irradiation with a red laser (632 nm) at powers ranging from 100 to 800 mW/cm2 for 10 or 20 minutes following the injection of the photosensitizing dye Photofrin II. Multiple cerebral arterioles were occluded by platelet aggregates containing frequent erythrocytes and leukocytes, identical to the thrombotic material in the carotid artery but different from the platelet aggregates seen in the carotid artery and the brain in the rose bengal model. Eighty infarcts were distributed randomly throughout the brain ipsilateral to the nonocclusive carotid thrombus. Significant heating (0.5 degree C or more) of the blood occurred only with laser powers higher (1,600 mW/cm2) or laser irradiations longer (25 minutes) than those used in the improved model of embolic stroke. This model mimics one mechanism of stroke in humans and provides a means to study systematically the morphological evolution of small cerebral infarcts.     

Singlet oxygen induced cerebral vasospasm: an experimental study in rats

A new experimental model is described which can be used as an alternative to study the effects of subarachnoid hemorrhage (SAH) in rats. Vasospasm of the basilar artery is induced photochemically after transpalatal illumination of intracisternally injected rose bengal in two different rat strains. Singlet oxygen, generated in the subarachnoid space, elicits vasospasm which has been demonstrated angiographically at 90 min and 24 h after photosensitisation. Sprague Dawley rats responded better than Wistar rats. Dilution of rose bengal in water was more vasospastic than dilution of rose bengal in artificial CSF. Since the action of singlet oxygen is similar to that of free radicals, this experiment gives an argument for the hypothesis that free radicals play a leading role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage.     

Embolic stroke from a carotid arterial source in the rat: pathology and clinical implications

We developed a new animal model of stroke which resembles human stroke more closely than existing models. We described the pathology produced in the brain following platelet embolism, previously described only in the retina. The common carotid artery of the rat was irradiated for 6.5 minutes with an argon laser at 514.5 nm after intravenous injection of a photosensitizing agent, rose bengal. A retinal embolus was seen in 1 rat 5 minutes after irradiation. A nonocclusive platelet thrombus was present in the carotid artery 50 minutes after irradiation, with almost all the platelet thrombus being cleared 24 hours later. Acute (1 to 10 days) changes in the brain included 44 small infarcts in 12/13 rats, cortical arterioles occluded with platelets and thickening of small vessels in normotensive rats. Chronic (4 to 12 weeks) changes included lacunes in the brains of normotensive rats and intimal proliferation of smooth muscle in the carotid artery. This is the 1st animal model of (1) stroke with emboli produced in vivo rather than injected into the carotid, (2) intimal proliferation of smooth muscle without invasion of the vessel, and (3) lacunes. This model provides results important to the laboratory study of stroke.     

The role of thromboxane (TX) A2 in rabbit arterial thrombosis induced by endothelial damage

To clarify the role of thromboxane (TX) A2 in arterial thrombus formation, we examined the antithrombotic effects of both a TXA2 synthetase inhibitor (CV-4151) and a TXA2 receptor antagonist (AA-2414) on the rabbit common carotid artery thrombosis which was induced by injury of the endothelium by treatment with 0.25% pronase solution. CV-4151 (1,10 mg/kg, p.o.) and AA-2414 (10 mg/kg, p.o.) significantly inhibited thrombus formation. Furthermore, the combined use of CV-4151 and AA-2414 (0.1 mg/kg, p.o. each) significantly inhibited thrombus formation, though these drugs at the same doses had no effect when administered singly. The plasma level of 11-dehydro TXB2 increased significantly during thrombus formation, and CV-4151 (10 mg/kg) markedly inhibited this increase. There was a significant correlation between the in vivo antithrombotic effects of these drugs and their ex vivo inhibitory effects on arachidonic acid-induced platelet aggregation. The antithrombotic effect of CV-4151 also correlated significantly with its ability to inhibit the production of serum TXA2. These results show that TXA2 may play an important role in the thrombus formation in arterial thrombosis.     

The antithrombotic effect of aspirin and dipyridamole in relation to prostaglandin synthesis

The antithrombotic effect of aspirin (ASA) and dipyridamole (DIP) was evaluated in rabbits in which platelet thromboxane A2 (TXA2) and arterial prostacyclin (PGI2) were measured. An intracarotid cannula thrombosis model previously shown to be sensitive to antiplatelet agents was used. Prostaglandins were determined by radioimmunoassays for thromboxane B2 (TXB2) and 6-keto-prostaglandin PGF1 alpha, the stable metabolites of TXA2 and PGI2. In the aspirin-treated animals, reduction in thrombosis was seen only in rabbits which received a low-dose (1-2 mg/kg), and was related to a selective suppression of platelet TXA2. In contrast, higher doses of ASA (10 or 100 mg/kg), which suppressed both TXA2 and PGI2, were not associated with thrombus inhibition. DIP alone had a lesser antithrombotic effect which was augmented by low-dose ASA but not by high-dose ASA. It is concluded that 1) the antithrombotic effect of ASA in this animal model is dependent on selective TXA2 suppression; 2) ASA has no antithrombotic properties beyond its inhibition of prostaglandin synthesis by platelets; 3) selective suppression of TXA2 in vivo can be achieved in rabbits by a single dose of ASA but only over a narrow dose-range; 4) DIP may have an antithrombotic effect additive to that of low-dose ASA; 5) measurement of serum TXB2 may be used to determine the minimal ASA dose necessary to suppress TXA2 and therefore be most likely to spare PGI2.     

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 microgram/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.     

Intravenously and topically applied magnesium in the prevention of arterial thrombosis

Magnesium (Mg) has been shown to reduce platelet aggregation both in vitro and ex vivo, and this antiplatelet effect may be advantageous in the prevention of arterial thrombosis. Previous animal studies have shown an antithrombotic effect of Mg also in vivo, but mainly with higher Mg concentrations ( approximately 3.0-4.0 mM). The objectives of the present study were to evaluate the antithrombotic effect of (1) intravenous Mg at a lower and clinically more relevant concentration and (2) topically applied Mg. The study comprised 30 male rats, randomly assigned into 3 groups: (1) placebo group, (2) intravenous Mg group, and (3) topical Mg group. A thrombogenic lesion was established by making a standardised arteriotomy in the right femoral artery. The vessel was transilluminated and thrombus formation was visualised dynamically by in vivo microscopy and recorded on videotapes. Thrombus area was measured after ended experiment by computer-assisted image analysis. Intravenously administered Mg, elevating the S-Mg level to 2.2 mmol/L, significantly reduced the mean thrombus area (p<0.05) compared to the control group. Topically applied Mg significantly decreased the maximum thrombus area, without any increase in S-Mg level (p<0.05). The Mg-treated groups showed no increase in bleeding complications. A transient fall in blood pressure was seen in the systemic Mg group, but blood pressures were not significantly different between any of the groups at the end of the experiment.In conclusion, topically as well as intravenously infused Mg reduce arterial thrombus formation in this in vivo rat model without compromising haemostasis.     

Electrically induced arterial thrombosis model in the conscious rat

Thrombus formation was electrically induced in the left common carotid artery of rats with preoccluded both vertebral and right carotid arteries. Following thrombus induction, animals became unresponsive and lost their righting reflex. The loss of righting reflex was observed when cortical tissular pO2 reached 55 +/- 9% of its initial value. A similar decrease in pO2 was induced by mechanical occlusion of the left carotid artery. The delay between discontinuation of current and the loss of righting reflex was measured to indirectly evaluate occlusive thrombus formation. The capacity of various antithrombotic agents to delay the righting reflex loss was studied. Heparin, ticlopidine and the thromboxane-endoperoxides receptor blocker, BM 13177, exerted a significant activity, whereas acetylsalicylic acid and the thromboxane synthetase inhibitors, OKY O46 and dazmegrel, were inefficient.     

On the mechanism of thrombolytic action of thromboxane synthetase inhibitors

Using our in vivo model for studying drugs which prevent deposition of thrombi or dissipate thrombi formed in extra-corporeal circulation over a collagen strip superfused with arterial blood of anaesthetized and heparinized cats, we have found that dazoxiben--a thromboxane synthetase inhibitor--possesses not only antithrombotic but also thrombolytic potency in vivo (ED50 = 3.8 mg/kg i.v.). The thrombolytic potency of dazoxiben was antagonized by aspirin at a dose of 50 mg/kg i.v. Moreover, dazoxiben stimulated the generation of prostacyclin in isolated rat aortic slices incubated in platelet rich plasma, but not in platelet poor plasma. It is suggested that the thrombolytic potency of thromboxane synthetase inhibitors after their systemic administration is associated with the release of prostacyclin and/or prostacyclin-stable metabolites by the vascular endothelium owing to feeding of prostacyclin synthetase with prostaglandin endoperoxides accumulated in platelets following the inhibition of thromboxane synthetase.     

The role of platelet hyperfunction in thrombus formation in hyperlipidemia

The mechanism of thrombus formation in hyperlipidemia was studied. Attempts at artificial creation of an arterial thrombus in control rabbits stenosing the femoral artery by ligature were not successful unless ellagic acid was administered by injection. Howevere, in rabbits with hyperlipidemia, mere creation of stenosis in the femoral artery resulted in a high percentage of thrombus formation. In rabbits with hyperlipidemia, both thromboxane (Tx) A₂ biosynthesis in platelets and prostacyclin (PGI₂) biosynthesis in the aorta were increased and these changes were noted at the level of cyclooxygenase in the arachidonic acid metabolic pathway. Therefore, these results suggest that thrombi are likely to be formed in hyperlipidemia and that such thrombus formation is due largely to platelet hyperfunction.     

Effect of diabetes on photochemically induced thrombosis in femoral artery and platelet aggregation in rats.

Effect of diabetes on thrombogenesis was examined by using a thrombus model with photochemical reaction in the rat femoral artery. In streptozotocin-induced diabetic rats for 8 weeks, the formation of thrombus following endothelial injury was significantly slower than that in non-diabetic rats. Insulin treatment normalized the abnormality of thrombogenesis. Aggregation in washed platelets from rats with diabetes was enhanced. However, platelet aggregation in whole blood was reduced in diabetic rats, and plasma from diabetic rats attenuated platelet aggregation. These results suggest that plasma factor(s) and/or other blood cells modify the hyperaggregability of platelets per se in vivo in diabetic rats. Treatment with insulin improved the aggregation in whole blood and washed platelets. In conclusion, diabetes induces the prolongation of thrombogenesis in the rat femoral artery. Hypoaggregability of whole blood is likely to be partly involved in the abnormal thrombogenesis.     

YAG laser-induced reperfusion of photothrombotic middle cerebral artery occlusion in rats]

The laser-driven photochemical occlusion of middle cerebral artery(MCA) is much easier, and less traumatic than standard electrocautery or even clip methods, while the infarct size is fairly reproducible. This study aimed to establish the system for YAG laser-induced reperfusion of photothrombotic MCA occlusion. Male spontaneously hypertensive rats(5-7 months old, 350-450 g) were anesthetized with halothane, endotracheally intubated, and mechanically ventilated. The photosensitizing dye rose bengal(20 mg/kg body weight) was administered intravenously over 90 sec starting simultaneously with 3 min of krypton laser irradiation(568 nm, 20 mW). The irradiated middle cerebral artery was completely occluded by an intraluminal thrombus. A YAG laser operating at 355 nm(16 mW, 15 Hz) was focused with a cylindrical lens and positioned with a mirror onto the occluded distal MCA. This YAG laser irradiation for approximately 3 min caused reperfusion of the thrombosed distal MCA. We demonstrated a novel method of reperfusion in the photothrombotic MCA occlusion model. This reperfusion model should facilitate study of the therapeutic window for reversibility in thrombotic stroke.     

Antithrombotic effects of ticlopidine,acetylsalicylic acid and dipyridamole in vascular shunt model in rats

Antithrombotic action of three platelet aggregation inhibitors was studied in a vascular shunt model of rats in which occlusive white thrombus was formed in most of animals within 4 hr after a loopshaped polyethylene cannula was installed between the carotid artery and jugular vein. We also measured platelet aggregation in plasma and PGI₂-like activity of the arterial segment from the rats treated with these agents. Ticlopidine significantly prevented the thrombus formation at oral doses above 5 mg/kg in approximate proportion to its inhibitory effect on platelet aggregation. Acetylsalicylic acid had no antithrombotic effect despite of its potent anti-platelet action but it blocked generation of PGI₂-like substance by the carotid artery. Dipyridamole did not inhibit platelet aggregation but apparently increased the generation of PGI₂-like activity, and it showed a tendency to prevent the shunt thrombosis at a high dose. Thus, the thrombus formation in this shunt model appears to be affected by PGI2-generation by the vascular tissue as wall as platelet function.     

Antithrombotic effects of recombinant hirudin in experimental angioplasty and intravascular thrombolysis

The effect of recombinant desulphatohirudin CGP 39393 (rH) on arterial thrombus formation and especially on thrombotic reocclusion after experimental angioplasty as well as after thrombolysis was investigated in rabbits. In the femoral artery thrombi were induced after endothelial damage of the vessel wall by a balloon catheter and following stasis. After removing the thrombus by angioplasty or after lysing it by streptokinase reocclusion of the artery was observed within a relatively short period of time. Subcutaneous injection of rH reduced the incidence of both primary thrombus formation and reocclusion in dependence on the dose administered. After a dose of rH of 2 mg/kg s.c. arterial thrombus formation was completely prevented and after administering 4 mg/kg s.c. thrombotic reocclusion did also not occur. Comparative studies with heparin showed that similar antithrombotic effects were only achieved at doses of 12 mg heparin/kg s.c. The results obtained suggest a clear potential of rH for prevention of thrombotic reocclusion in clinical states.     

Antithrombotic effect of ticlopidine in a platelet-independent model of venous thrombosis

The antithrombotic activity of ticlopidine demonstrated in a variety of experimental models of thrombosis has been explained by its antiaggregating properties. This study describes the antithrombotic effect of ticlopidine in a platelet independent model of venous thrombosis. In the rat, ligature of the inferior vena cava induces thrombosis. Antiaggregating drugs (acetylsalicylic acid, dipyridamole, sulfinpyrazone) are inactive while anticoagulants (heparin, acenocoumarol) are highly antithrombotic. Ticlopidine reduces thrombus weight significantly and dose-dependently (ED 50 = 150 mg/kg/day X 3 days). Thrombocytopenia induced by injection of anti-platelet anti-serum was found not to modify thrombus formation. Yet, even in these conditions, ticlopidine remains active. Acetylsalicylic acid treatment does not prevent the antithrombotic effect of ticlopidine, indicating that its action is independent of PGI2 synthesis. These results demonstrate that ticlopidine acts as an antithrombotic agent in a venous thrombosis model in which platelets play a minor role.     

Effects of immuno-potent drugs and their association with an unfractionated heparin on an experimental venous thrombosis.

Immuno-potent drugs are largely used in human medicine. The aim of this study was to determine the role of two immuno-modulators (sodium diethyl-dithiocarbamate, RU 41 740) and two immuno-suppressors (methylprednisolone, cyclosporin A) alone or in association with an unfractionated heparin (Calciparin), on an experimental venous thrombosis made by vena cava ligation in male Wistar rats. Each immuno-potent drug was administered for six days before the thrombus induction at the same dosage (10mg/kg b.w.), and the Calciparin, used as treatment of the thrombosis, was administered two hours after the thrombus induction at the dose of 1mg/kg b.w. Immuno-treatment potentiated thrombus formation: the increase in thrombus weight was greater with immuno-modulators (43% on average in comparison with placebo) than with immuno-suppressors (20%). In association with Calciparin the antithrombotic effect was also potentiated and more marked with the immuno-modulators than with immuno-suppressors. An increase in circulating monocytes was observed in all groups whether Calciparin was present or not. Coagulation tests were not affected by immuno-therapy. However, immuno-modulators led to an inhibition of platelet aggregation. In conclusion, this trial seems to show a probable effect of immunological cells in thrombosis and in the antithrombotic effect of heparin, but the mechanism involved is not yet determined.     

Antithrombotic effect of SM-20302, a nonpeptide GPIIb/IIIa antagonist, in a photochemically induced thrombosis model in guinea pigs.

SM-20302, a synthetic inhibitor of the fibrinogen receptor of platelets, has been shown to inhibit the platelet aggregation induced by various stimuli. In the present study, we performed ex vivo platelet aggregation studies by using heparinized platelet-rich plasma (PRP) as well as citrated PRP and compared the antiaggregatory activity with the in vivo antithrombotic efficacy of SM-20302. The oral administration of SM-20302 (0.3-10 mg/kg) to guinea pigs completely inhibited the ADP-induced ex vivo platelet aggregation in citrated PRP. In heparinized PRP, SM-20302 (1-10 mg/kg) showed a dose-dependent inhibition of ex vivo platelet aggregation, and it exhibited complete inhibition at a dose of 3 and 10 mg/kg, respectively. The concentration of ionized calcium in the citrated samples was approximately 35 times lower than that in heparinized samples. Chelation of ionized calcium caused an enhancement of the antiaggregatory activity of SM-20302 in guinea pig heparinized PRP in vitro. And addition of CaCl2 to citrated PRP reversed the enhancement. Citrate therefore appeared to enhance the inhibitory activity of SM-20302 by lowering the ionized calcium levels. We also examined the in vivo efficacy of SM-20302 in a photochemically induced femoral artery thrombosis model in guinea pigs. The photochemical injury of the endothelium of femoral artery resulted in a progressive decline in the blood flow. The oral administration of SM-20302 (0.1-3 mg/kg) produced a dose-dependent maintenance of the femoral artery patency and significantly prolonged the time to occlusive thrombus formation at a dose of 1 and 3 mg/kg, respectively. These results suggest that SM-20302 may be an orally active antithrombotic agent, and its in vivo antithrombotic efficacy appeared to correlate well with the ex vivo platelet inhibition in PRP prepared from heparinized blood but not in PRP anticoagulated with citrate.