Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.

BACKGROUND: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. METHODS: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. FINDINGS: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. INTERPRETATION: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. FUNDING: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).

[Study on prevention effect of huoluotongnao tablet on stroke (I)].

OBJECTIVE: To study the prevention effect of Huoluotongnao tablet on stroke. METHODS: Thrombosis on arteriovenous shunt rats model, platelet aggregation and hypertension combined high cholesterol rats model were used. RESULTS: Huoluotongnao tablet high and low dosage could inhibit the formation of arteriovenous thrombosis and platelet aggregation significantly ,the inhibition rate was 17.71%, 22.69%, 20.34% and 24.43%, respectively. Pretreatment of Huoluotongnao tablet could inhibit the formation of arteriovenous thrombosis significantly; The levels of CHOz in all treatment groups of hypertension combined high cholesterol rats model were decreased significantly,the levels of TGz and LDL-C were decreased in the high dosage group,the blood pressure was decreased in the middle dosage group. eta bL, eta P and eta r (B/P) were decreased in the middle and high dosage groups. eta bM, AI and CY were decreased in the middle and high dosage groups. Huoluotongnao tablet had effect on blood lipid,blood pressure and hemorheology and in a dose-dependence manner. Its minimal effecting dose was the middle dose. g/kg (crude drug) and has certain prevention effect on stroke. CONCLUSION: Huoluotongnao tablet's minimal effecting dose is 1.28

[Study of dahuangzhechong pills on anti-arterial thrombosis with the orthogonal design].

OBJECTIVE: To screen the main component of Dahuangzhechong pill's anti-arterial thrombosis with the orthogonal design and refine Dahuangzhechong pills. METHODS: In accordance with the orthogonal design table (L(16)2(15)), divided herbs into 16 groups and made the appropriate liquid. The liquid was gave to SD rats by intragastric administration,the model group, normal control group received the same volume of physiological saline. Isolated rats' carotid artery after intragastric administration a week,modeled according to ferric chloride inducement the carotid artery thrombosis method, then collected blood, detected content of platelet, thromboxane B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), sheared and measured dry weight of the modeling artery, then placed arteries in 10% formalin fixation, observed morphological changes in vascular tissue by HE staining. RESULTS: Pathological examination revealed: each experimental group had thrombosis, softening, dissolution, absorption, and intimal injury, but the severity of thrombosis were diferent. Orthogonal analysis showed: 1, influence on dry weight of thrombus: rhubarb, ground beetle, leeches, peach seed, dry paint, except dry paint P<0.05, the others P<0.01.2, influence on plasma 6- keto-PGF1alpha level: peach seed, dry paint, ground beetle, gadfly, grubs, leeches, rhubarb, except rhubarb P<0.05, the others P<0.01.3, influence on plasma TXB2: ground beetle, peach seed, dried paint, rhubarb, leeches, except leech P<0.05, the others P<0.01.4, influence on platelet count: peach seed, dry paint, rhubarb, ground beetle, gadfly, leeches, except gadfly, leeches P<0.05, the others P<0.01. CONCLUSION: Anti-artery thrombosis of Dahuangzhechong Pill is most closely related with rhubarb, ground beetle, leeches, peach seed, dry paint and gadfly.

Dietary α-linolenic acid inhibits arterial thrombus formation, tissue factor expression, and platelet activation.

OBJECTIVE: Plant-derived α-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown. METHODS AND RESULTS: Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-κB activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells. CONCLUSIONS: ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.

[Effect of Xuesaitong drop pills on experimerntal thrombosis and thrombolysis in rats].

OBJECTIVE: To study the effect and the mechanism of Xuesaitong drop pills (total saponins in Radix Notoginseng; XDP) on experimental thrombosis, thrombolysis and blood theology. METHOD: First, the rats were randomly divided into five groups: control, XDP (90, 30, 10 mg x kg(-1)), Xuesaitong tablet (XP) 30 mg x kg(-1). Then the effect of the drugs on thrombus and thrombosis was studied after the ratsthrombosis was induced by the arteriovenous shunt. Second, the rats were randomly divided into seven groups: model, XDP (90, 30, 10 mg x kg(-1)), XT (90, 30 mg x kg(-1)), lumbrukinase capsule. Then the effect of the drugs on thrombus and thrombosis was studied after the rats'thrombosis was induced by the electrical stimulation of common carotid artery. Third, the rats were randomly divided into six groups: control, model, XDP (80, 40 mg x kg(-1)), XT (40, 20 mg x kg(-1)). Then the effect of the drugs on blood circulation promoting was observed after the rats'acute blood stasis induced by adrenalin and icy water. RESULT: XDP 90, 30 mg x kg(-1) could notably lighten the wet-weight and dry-weight of thrombus in the arteriovenous shunt model in rats in a dose-dependent manner (P < 0.01). XDP 90 mg x kg(-1) with intragastric administration for 3 days had the satisfactory effect on thrombolysis after the rat's thrombosis was induced by the electrical stimulation of common carotid artery (P < 0.01). XDP 80, 40 , 20 mg x kg(-1) reduced significantly erythrocyte aggregation (P < 0.01) and decreased the whole blood viscosity at low shear rate (P < 0.05). XDP 80, 40 mg x kg(-1) reduced the whole blood viscosity at high shear rate and plasma viscosity (P < 0.05). XDP 80 mg x kg(-1) decreased the whole blood viscosity at high shear rate (P < 0.05). CONCLUSION: XDP can significantly inhibit the thrombosis and has the satisfactory effect on thrombolysis. One kind of the mechanism is related to the effect on blood rheology.

Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor.

BACKGROUND/OBJECTIVE: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. In the present study, we describe the development of a murine model of vena cava thrombosis and its use to characterize the antithrombotic activity of potato carboxypeptidase inhibitor (PCI) of TAFIa (activated TAFI) in mice. METHODS/RESULTS: Vena cava thrombosis was induced by various concentrations of FeCl(3) in C57BL/6 mice. A relatively mild stimulus (3.5% FeCl(3)) induced thrombosis that was consistent and sensitive to reference antithrombotic agents such as clopidogrel and heparin. Dose-response studies identified a PCI dose (5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1), i.v.) that produced a maximum 45% decrease in vena cava thrombus mass as assessed by protein content (n = 8, P < 0.01 compared to vehicle) in the 3.5% FeCl(3)-induced model without exogenous tissue plasminogen activator administration. In contrast, PCI had no effect on 3.5% FeCl(3)-induced carotid artery thrombosis in mice. In a tail transection bleeding model, the 5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1) dose of PCI increased tail-bleeding time up to 3.5 times control (n = 8, P < 0.05). The ex vivo activity of antithrombotic doses of PCI was also demonstrated by the enhanced lysis of whole blood clots formed in a thrombelastograph with the addition of a sub-threshold concentration of tPA. CONCLUSION: These studies provide evidence for a role of TAFIa in venous thrombosis in mice, and describe an optimized vena cava injury model appropriate for the evaluation of antithrombotic drugs and the characterization of novel therapeutic targets.

Effects of total flavone of Abelmoschl Manihot L. Medic on the function of platelets and its mechanism.

OBJECTIVE: To study the effects of total flavone of Abelmoschl Manihot L. Medic (TFA) on the function of platelets and to explore its mechanism. METHODS: Rat models of artery-veins bypassing thrombus formation were used. The platelets of rabbits were collected. Platelet aggregation was induced by collagen and intracellular calcium ion concentration ([Ca(2+)]i) was assayed by Fura-2 method. RESULTS: TFA (25, 50, 100 mg/kg) significantly and dose-dependently reduced the weight of thrombus. TFA (0.025, 0.05, 0.1 mg/ml) possessed dose-dependant inhibitory effects on rabbits' platelet aggregation induced by collagen. TFA significantly reduced the resting and CaCl(2)-induced increase of free intracellular calcium concentration ([Ca(2+)]i) in rabbit platelet in vitro. CONCLUSION: TFA has an antiplatelet effect via the inhibition on the influx of Ca(2+).

Ethanol plus caffeine (caffeinol) for treatment of ischemic stroke: preclinical experience.

BACKGROUND AND PURPOSE: Ethanol and caffeine are 2 common psychoactive dietary components. We have recently shown that low-dose ethanol plus caffeine results in a 70% to 80% reduction of infarct volume after reversible common carotid/middle cerebral artery (CCA/MCA) occlusion in rats. The combination (caffeinol) was effective after either oral pretreatment or intravenous administration starting up to 2 hours after stroke onset. Ethanol alone aggravated ischemic damage, while caffeine alone was without effect. Daily caffeinol for 2 weeks before ischemia eliminated the neuroprotection seen with acute treatment (tolerance). The purpose of our present study was to further characterize the properties of caffeinol as a possible treatment for ischemic stroke. METHODS: The transient CCA/MCA occlusion model was used in all experiments. Five sets of experiments were conducted (1) to test the effectiveness of various doses of ethanol (0.2 to 0.65 g/kg) and caffeine (3 to 10 mg/kg) in the caffeinol mixture; (2) to test whether the neuroprotective dose of caffeinol can improve behavioral dysfunction; (3) to test whether chronic ethanol or caffeine before ischemia will affect efficacy of caffeinol treatment; (4) to test whether the protective effect of caffeinol can be improved by pairing it with 35 degrees C hypothermia; and (5) to test whether caffeinol affects frequency of hemorrhage after administration of recombinant tissue plasminogen activator (rtPA) in ischemic animals. RESULTS: Doses as low as 0.2 g/kg of ethanol and 6 mg/kg of caffeine in the caffeinol were effective in reducing cortical infarct volume and behavioral dysfunction after transient CCA/MCA occlusion. Daily exposure to ethanol but not caffeine before CCA/MCA occlusion eliminated the therapeutic efficacy of acute caffeinol treatment, similar to the tolerance observed after chronic exposure to caffeinol. The therapeutic effect of caffeinol could be further improved by pairing it with mild intraischemic hypothermia, and caffeinol did not increase hemorrhagic infarction when given in combination with rtPA. CONCLUSIONS: Low doses of caffeinol, equivalent to no more than 2 to 3 cups of strong coffee and 1 cocktail, are consistently and highly neuroprotective, are well tolerated, can be added to other therapies to increase the effect of each, and do not interfere with or complicate rtPA therapy. Caffeinol is an appropriate candidate for clinical trial in stroke patients, although it may be less effective in patients with regular alcohol intake.

Correlation between the in vivo efficacy of GPIIb/IIIa receptor antagonists (m7E3, MK-383 and DMP-728) and ex vivo platelet inhibition.

In vivo antithrombotic efficacy of GPIIb/IIIa receptor antagonists (m7E3, MK-383 and DMP-728) was studied with respect to their ex vivo platelet inhibition in heparinized platelet-rich plasma (hPRP) and citrated platelet-rich plasma (cPRP) using a canine model of carotid artery thrombosis. For each drug group (n = 6), the right carotid artery was used as control vessel and resulting occlusive thrombus was kept in situ to examine the direct thrombolytic efficacy of the antagonists. Thirty minutes after occlusion of control vessel, a low or high dose of each antagonist was administered and the left carotid artery was used as test vessel. All control vessels occluded within 86-96 min in response to electrolytic injury. The incidence of occlusion with lower doses of m7E3, DMP-728, and MK-383 was 100, 33 and 100%, respectively; corresponding times to occlusion were 174, 220 and 118 min. Lower doses inhibited ADP- or AA-induced platelet aggregation in cPRP (>80%). Incidence of occlusion with high doses of m7E3, DMP-728 and MK-383 was 33, 0 and 0%, respectively; corresponding times to occlusion were 209, >240 and >240 min. Higher doses inhibited aggregation in cPRP (>80%), but only partially in hPRP (45-66%). Dose-dependent prolongation of bleeding time occurred with all antagonists. None of the antagonists lyzed preformed thrombi in control vessels. The results indicate that ex vivo platelet aggregation conducted in hPRP, as opposed to conventional cPRP, provides a better assessment of the in vivo efficacy of GPIIb/IIIa receptor antagonists.

Prevention of arterial thrombosis using a novel heparin with enhanced antiplatelet activity and reduced anticoagulant activity.

PURPOSE: Thrombosis after arterial injury is often initiated by von Willebrand factor (vWF)-dependent platelet accumulation. A promising antithrombotic strategy is the interruption of platelet/vWF interactions. Previously, we demonstrated how chemical and affinity modification can enhance heparin's anti-vWF activity while reducing conventional anticoagulation. Here, we investigated whether a modified heparin can block platelet-dominated arterial thrombosis. METHODS: Standard heparin was oxidized with periodate, refined to have high vWF affinity and inhibitory potency, and tested in a guinea pig model of platelet-dependent arterial thrombosis. In this model, a controlled mechanical arterial injury yields cyclic flow variations (CFVs) caused by recurrent accumulation of platelet thrombi. RESULTS: All six control animals developed CFVs (mean, 10.4 +/- 2.6 CFVs), and six of seven animals treated with standard heparin also developed CFVs (mean, 7.6 +/- 4.6). Only one of six animals treated with the anti-vWF heparin and one of six treated with AJvW-2 (an anti-vWF antibody) developed CFVs (mean, 2.0 +/- 4.9 and 0.5 +/- 1.2, respectively). Thus both the modified heparin and AJvW-2 were more effective than standard heparin (p < 0.03). Bleeding times and platelet counts were unaffected. A modified activated partial thromboplastin time was less prolonged by the modified high-affinity heparin (91 +/- 17) seconds) than by standard heparin (144 +/- 30 seconds; p < 0.01). CONCLUSIONS: The modified heparin with high vWF affinity was a more effective arterial antithrombotic agent, with fewer conventional anticoagulant effects than standard heparin. Interruption of the vWF/platelet interaction is a promising antithrombotic strategy that may be met by novel heparin-based antithrombotic drugs.

[Effect of Ganoderma japonicum (Fr.) Lloyd mixture on experimental thrombosis].

The therapeutic effect of Ganoderma japonicum(Fr.) Lloyd mixture on thrombosis and its mechanism were studied. The results showed that Ganoderma japonicum(Fr.) Lloyd mixture inhibited thrombus formation in vitro and in vivo, the thrombus weight and length formed in the rabbit common carotid artery and external jugular vein were significantly decreased in the experimental group compared with the control (P < 0.01). The results suggest that Ganoderma japonicum(Fr.) Lloyed mixture has anti-thrombotic effect, blood coagulation and platelet activation were inhibited, and the ability of vascular endothelial cells against the process of thrombosis was enhanced.

Prevention of early reocclusion after thrombolysis of copper coil-induced thrombi in the canine carotid artery: comparison of PEG-hirudin and unfractionated heparin.

Cofactor-independent thrombin inhibitors as adjunctive treatment to thrombolysis have been found to enhance reperfusion and reduce the incidence of early reocclusion more effectively than heparins. However, all thrombin inhibitors presently available are rapidly cleared from the circulation which may cause rebound effects after cessation of treatment. To evaluate the effect of PEG-hirudin (LU 87981) a new, long acting derivative of hirudin as adjunctive treatment to rt-PA, a thrombotic occlusion of the carotid artery was induced in mongrel dogs by means of a copper coil. Vessel patency was continuously monitored with an electromagnetic flow probe. Thrombolysis of the occluded artery was induced by administration of 40 micrograms x kg-1 + 240 micrograms x kg-1 x h-1 rt-PA (low dose) or 80 micrograms x kg-1 + 480 micrograms x kg-1 x h1 rt-PA (high dose). With high dose rt-PA treatment, patency was achieved in all animals within 50 min (range 24 to 75), with low dose rt-PA treatment only in 6 out of 8 animals after 73 min (range 26 to 117). Concomitant administration of PEG-hirudin (0.3 mg x kg-1 bolus + 0.15 mg x kg-1 x h-1 infusion) increased the incidence of reperfusion in the low dose rt-PA group to 100% while the reperfusion time was shortened from 73 min in the corresponding control group to 38 min (range 20 to 75 min) in the group given PEG-hirudin (p = 0.065, Mann-Whitney U-test). The carotid artery blood flow, which rapidly declined to zero within 18 to 27 min after discontinuing low or high dose rt-PA infusions remained at a sustained level for the whole observation period of 4 h only in the group given PEG-hirudin. Only one animal reoccluded after 229 min. Unfractionated heparin (UFH) given at a dose of 0.3 mg x kg-1 bolus + 0.3 mg x kg-1 x h-1 infusion did not improve the incidence of reperfusion or lower the incidence of reocclusion. Buccal bleeding time was prolonged after high dose rt-PA treatment and after low dose rt-PA with adjunctive UFH- or PEG-hirudin treatment. Buccal blood loss was not significantly affected by either treatment. In conclusion, these experiments indicate that early reocclusion after thrombolysis can effectively be diminished by concomitant treatment with the long acting thrombin inhibitor PEG-hirudin with moderate effects on bleeding time and aPTT.

[Inactivated factor VII exercises a powerful antithrombotic activity in an experimental model of recurrent arterial thrombosis]. / Il fattore VII inattivato esercita una potente attività antitrombotica in un modello sperimentale di trombosi arteriosa ricorrente.

The extrinsic coagulation pathway is activated when tissue factor (TF) is exposed as a consequence of arterial damage. TF binds to factor VII (FVII) or activated FVII (FVIIa), generating a complex that activates both FX and FIX, ultimately leading to thrombin formation. To determine whether inhibition of FVII binding to TF would result in antithrombotic effects, active site-blocked FVIIa (FVIIai) was used in a rabbit model of intravascular thrombus formation. In addition, to study the interaction between extrinsic coagulation pathway activation and platelet aggregation, in the same model of intravascular thrombus formation, recombinant human FVIIa was administered in antiplatelet-treated rabbits. Cyclic flow variations (CFVs), due to recurrent thrombus formation, were initiated by placing an external constrictor around the endothelially-injured rabbit carotid arteries (Folt's model). Carotid blood flow was measured continuously by a Doppler flow probe placed proximally to the constrictor. CFVs were induced in 29 New Zealand White rabbits. After CFVs were observed for 30 min, the animals were randomly divided in four groups: 5 animals received via a small catheter (26G) placed proximally to the stenosis, an intra-arterial infusion of human recombinant FVIIai (0.1 mg/kg/min for 10 min); 9 animals received AP-1, a monoclonal antibody against rabbit TF (0.1 mg/kg i.v. bolus); 7 animals received ridogrel, a dual thromboxane A2 synthetase inhibitor and thromboxane A2 receptor antagonist (10 mg/kg i.v. bolus); finally, 8 rabbits received aurintrycarboxilic acid (ATA), an inhibitor of platelet glycoprotein Ib/von Willebrand factor interaction (10 mg/kg i.v. bolus). FVIIai abolished CFVs in 5 of 5 animals (CFV frequency minutes 0 cycles/hour; p < 0.05; carotid blood flow velocity minutes 106 +/- 9% of the baseline values; NS vs baseline). AP-1 abolished CFVs in 7 of 9 animals (CFV frequency minutes 0 cycles/hour; p < 0.05; carotid blood flow velocity minutes 58 +/- 35% of the baseline values; NS vs baseline). Finally, in all the animals receiving ridogrel or ATA CFVs were abolished (CFV frequency 0 cycles/hour; p < 0.05 in both groups; carotid blood flow velocity, respectively 62 +/- 32 and 66 +/- 40% of the baseline values; NS vs baseline in both groups). Thirty minutes following inhibition of CFVs, in the FVIIai treated rabbits, human recombinant FVIIa was infused, via the small catheter placed proximally to the stenosis, at the dose of 0.1 mg/kg/min for 10 min. In the other three groups, FVIIa, at the same dose, was infused i.v. Infusion of FVIIa restored CFVs in all FVIIai treated animals and in 6 of 7 AP-1 treated animals, thus indicating that AP-1 and FVIIai bindings to TF was competitive and was replaced by FVIIa. Infusion of FVIIa failed to restore CFVs in ridogrel e ATA treated rabbits (1 of 7 and 0 of 8 rabbits, respectively), showing that activation of extrinsic coagulation by FVIIa was overcome by inhibition of platelet function. Activated partial thromboplastin time, and ex vivo platelet aggregation in response to ADP and thrombin, were not different after FVIIai infusion, while prothrombin time was slightly but significantly prolonged as compared to baseline values. Thus, FVII-VIIa plays an important role in initiating thrombus formation in vivo. Administration of FVIIai exerts a potent antithrombotic effects in this model without affecting systemic coagulation. In addition, in this model platelets exert an important role in arterial thrombosis, since in the presence of inhibition of platelet function, activation of the extrinsic coagulation pathway failed to restore thrombus formation.

Short-term and long-term role of platelet activating factor as a mediator of in vivo platelet aggregation.

BACKGROUND: Platelet activating factor (PAF) is a phospholipid released upon stimulation by a variety of cells and has been implicated in several pathophysiological events such as asthma and inflammatory diseases. However, although the ability to aggregate platelets in vitro was the first biological activity ascribed to PAF, its role in contributing to the in vivo formation of arterial thrombi has not been thoroughly clarified. METHODS AND RESULTS: Intravascular platelet aggregation was initiated in two different animal models of arterial stenosis and endothelial injury. An external constrictor was positioned around rabbit carotid arteries and canine coronary arteries. After placement of the constrictor, a typical pattern of flow developed in the stenotic vessels. This pattern of flow, characterized by progressive reductions of carotid or coronary blood flow followed by spontaneous or induced restorations of flow (cyclic flow variations, CFVs), is related to recurrent platelet aggregation at the site of the stenosis followed by dislodgment of the thrombus. After observing CFVs for 30 minutes, BN52021 (up to 1.2 mg/kg), a potent and selective PAF antagonist, was given intravenously to rabbits (n = 12) and dogs (n = 10). BN52021 completely inhibited CFVs in 10 of 12 rabbits, whereas it was relatively ineffective in abolishing CFVs in dogs (only 2 of 10 animals inhibited). This different effect of BN52021 was not explained by too small a dose of the drug to achieve a complete blockade of PAF receptors in dogs, since ex vivo platelet aggregation was completely inhibited in both rabbits and dogs in response to exogenous PAF at concentrations up to 10(-5) mol/L. In a second group of 10 dogs, the hypothesis that PAF may become an important mediator of CFVs in dogs only several hours after endothelial injury was tested. After 30 minutes of baseline CFVs, these animals received a bolus of BN52021 up to 1.2 mg/kg. After this treatment, CFVs were completely abolished in 2 of 10 animals. The remaining 8 dogs were followed for an additional 8-hour period, at the end of which a second bolus of BN52021 was given. At this time, BN52021 was effective, as CFVs were abolished in 6 of 8 animals. These effects of BN52021 at 8 hours were not the consequence of a cumulative dose of the compound, since ex vivo platelet aggregation in response to PAF returned to baseline values immediately before administering the second dose. To identify possible sources of PAF other than aggregating platelets at the site of arterial stenosis, dogs in a third group were killed after 30 minutes (n = 7) and after 8 hours (n = 8) of CFVs. Histological sections of the stenotic coronary artery showed a marked leukocyte infiltration in these arterial segments after 8 hours of CFVs, whereas sections from dogs killed after 30 minutes showed only moderate or no infiltration. CONCLUSIONS: These data demonstrate that PAF plays an important role as a mediator of platelet aggregation in vivo in rabbits and dogs. In the canine model, PAF appears to become more important after leukocyte infiltration of the arterial wall, as it may contribute to initiating enough platelet activation to lead to cyclic flow variations at sites of arterial stenosis and endothelial injury. Data from the present study suggest that PAF antagonists may be used as antiplatelet agents.

Interruption of vascular thrombosis by bolus anti-platelet glycoprotein IIb/IIIa monoclonal antibodies in baboons.

PURPOSE: Because the platelet membrane receptor glycoprotein IIb/IIIa plays a central role in the recruitment of platelets into forming thrombus, therapeutic inhibition of this receptor complex may be particularly useful to prevent thrombosis after small vessel arterial manipulation. METHODS: The relative hemostatic safety and antithrombotic efficacy for thrombus formation at sites of endarterectomy and implanted prosthetic vascular graft of a murine monoclonal antibody (LJ-CP8) against platelet glycoprotein IIb/IIIa have been determined in baboons after bolus injections in doses (10 mg/kg) that block platelet receptor function for fibrinogen and other adhesive glycoproteins (absent platelet aggregation and bleeding times > 30 minutes without affecting circulating platelet counts). RESULTS: Thrombus formation was eliminated by LJ-CP8 at sites of surgical endarterectomy in fresh segments of homologous aorta incorporated into chronic exteriorized arteriovenous femoral shunts (accumulation of indium 111-labeled platelets fell from 4.40 +/- 0.89 x 10(9) platelets/cm in control animals [n = 6] to 0.23 +/- 0.01 x 10(9) platelets/cm in treated animals [n = 4]; p < 0.005). The formation of thrombus was also abolished by LJ-CP8 at sites of 1 cm prosthetic vascular grafts (4 mm inner diameter polytetrafluoroethylene grafts) interposed into common carotid arteries (deposition of indium 111-labeled platelets decreased from 2.57 +/- 0.43 x 10(9) platelets/cm [n = 5] to 0.16 +/- 0.06 x 10(9) platelets/cm, [n = 4]; p = 0.004). However, LJ-CP8 injections produced substantial bleeding in the surgical wound during the first few hours after operation. Thirty days after operation all four graft implants were patent in JJ-CP8-treated animals compared with two of five in control animals (p = 0.06). CONCLUSIONS: We conclude that profound inhibition of platelet glycoprotein IIb/IIIa receptor function by single bolus injection of LJ-CP8 monoclonal antibody transiently abolishes platelet hemostatic function, eliminates acute thrombus formation at sites of endarterectomy and prosthetic vascular graft implants, and may improve vascular patency.