Andrew B. Kelly

Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 125I-fibrinogen accumulation. Intravenous infusion of alpha-thrombin, 1-2 U/kg-min for 1 h, increased baseline activated PC levels (approximately 5 ng/ml) to 250-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by > 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin D-dimer plasma levels (P < 0.01), but did not affect bleeding times. Thrombins antithrombotic effects were blocked by infusing a monoclonal antibody (HPC-4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy.

Induction of Vascular Endothelial Growth Factor in Balloon-Injured Baboon Arteries: A Novel Role for Reactive Oxygen Species in Atherosclerosis

Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H2O2. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 mumol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 mumol/L H2O2. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 mumol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.

Clopidogrel Inhibition of Stent, Graft, and Vascular Thrombogenesis With Antithrombotic Enhancement by Aspirin in Nonhuman Primates

BACKGROUND A recent study showed that clopidogrel reduces thrombo-occlusive complications in patients with symptomatic atherosclerosis more effectively than aspirin. METHODS AND RESULTS The effects of clopidogrel and aspirin have been compared, singly and in combination, for measurements of 111In-labeled platelets and 125I-labeled fibrin deposition in baboon models of arterial thrombosis and related to platelet aggregation and expression of activation epitopes induced by ADP, collagen, and thrombin receptor agonist peptide (TRAP) and to template bleeding times (BTs). Low-dose oral clopidogrel (0.2 mg. kg-1. d-1) produced cumulative (1) intermediate decreases in 111In-platelet and 125I-fibrin deposition for segments of prosthetic vascular graft, deployed endovascular metallic stents, and endarterectomized aorta (P<0.009 in all cases); (2) elimination of ADP-induced platelet aggregation (P<0.001); (3) modest inhibition of collagen-induced platelet aggregation (P<0.01); (4) no reduction in TRAP-induced platelet aggregation; and (5) minimal prolongation of BTs (P=0.03). High-dose oral clopidogrel (>/=2 mg/kg) produced the same effects within 3 hours. The effects of clopidogrel dissipated over 5 to 6 days. Aspirin 10 mg. kg-1. d-1 alone did not decrease 111In-platelet and 125I-fibrin deposition on segments of vascular graft but detectably decreased 111In-platelet and 125I-fibrin accumulation on stents (P<0.01), minimally inhibited ADP- and collagen-induced platelet aggregation (P<0.05 in both cases), and minimally prolonged BTs (P=0.004). Within 3 hours of aspirin administration, the antithrombotic effects of acute high-dose or chronic low-dose clopidogrel were substantially enhanced, and BTs were modestly prolonged without inhibiting platelet aggregation induced by TRAP (P<0.001 in all cases compared with clopidogrel alone). CONCLUSIONS Clopidogrel produces irreversible, dose-dependent, intermediate reduction in thrombosis that is substantially enhanced by the addition of aspirin. The effects of combining aspirin and clopidogrel need to be evaluated in patients at risk of vascular thrombosis.

Inhibition of thrombus formation by activated recombinant protein C in a primate model of arterial thrombosis.

Activated protein C (APC) is an antithrombotic enzyme. The therapeutic potential of infused human recombinant APC (rAPC) was studied in a primate model of platelet-dependent thrombosis. Eight baboons with chronic femoral arteriovenous shunts received rAPC infusions for 1 hour. The shunts were extended with 5-cm long, 4-mm-i.d. thrombogenic Dacron graft segments for the time of infusion. The plasma level of the enzyme, the blood flow in the shunt, and the deposition of indium-111-labeled platelets and iodine-125 fibrinogen on the graft were measured. The influence of rAPC infused at doses of 0.25 and 1.0 mg/kg-hr was compared with the effects of control infusions of saline. Five of eight control grafts occluded within 60 minutes, whereas there was no change in the blood flow during rAPC infusion. Deposition of platelets was inhibited by 13 +/- 10% and by 42 +/- 13% (mean +/- SEM) after 30 minutes of infusion at the two doses, which gave rise to circulating rAPC plasma concentrations of 0.4 and 1.9 mg/l, respectively. Both doses significantly inhibited fibrin deposition in the graft. Circulating plasma markers of thrombus formation and of fibrinolysis did not increase significantly during rAPC infusion; measurements of bleeding time were also within normal limits. Thus, rAPC, like human plasma-derived APC, inhibited thrombus formation without impairing primary hemostasis.

Interruption of vascular thrombus formation and vascular lesion formation by dietary n-3 fatty acids in fish oil in nonhuman primates.

BackgroundBecause of discrepant claims regarding the relative biological effects of n-3 fatty acids (n-3FAs), we have concurrently measured the effects of dietary n-3FAs on blood and vascular lipid composition, hemostatic function, blood thrombotic responses, vascular thrombus formation, and vascular lesion formation in baboons. Methods and ResultsDietary n-3FAs displaced n-6FAs in plasma, platelets, blood vessels, and corresponding urinary eicosanoid metabolites (p<0.01 in all cases) within weeks after initiation of a semipurified diet containing 1 g/kg per day n-3FA-ethyl ester concentrate (composed of two thirds eicosapentanoic acid and one third docosahexanoic acid). Coincidentally, platelet hemostatic function became minimally impaired (template bleeding times prolonged from 4.3±0.5 minutes to 7.6±+1.3 minutes, p=0.039); concentrations of collagen producing half-maximal platelet aggregation increased (from 6.4±2.1 to 8.5±2.5 μg/mL, p=0.045); and tissue factor expression by endotoxin-stimulated blood monocytes fell (from 6.5+1.2 to 1.7±0.14 mU/106 cells, p<0.005). Dietary n-3FAs decreased deposition of platelets onto thrombogenic segments of Dacron vascular graft incorporated into chronic exteriorized femoral arteriovenous (AV) shunts, a thrombotic process resistant to the effects of both aspirin and heparin (111In-labeled platelet deposition decreased from 14.1+1.4x 109 platelets/5-cm segment at 40-60 minutes with occlusion to 7.5±0.8x109 platelets/S-cm segment without occlusion; p<0.001). Platelet deposition onto segments of endarterectomized homologous normal aorta in the AV shunts of n-3FAtreated animals was similarly reduced (from 4.4±0.9 to 1.8±0.4x 109 platelets; p<0.01). Dietary n-3FAs interrupted vascular thrombus formation at sites ofsurgical carotid endarterectomy (platelet deposition, 1.5±0.4 versus4.4±+.Ox109 platelets in untreated controls; p<0.001). Moreover, endarterectomized aortic segments (EASs) from n-3FA-treated donors exhibited little capacity to induce thrombus formation when tested in the AV shunts of control recipient animals (0.24±0.10 versus 4.4±0.90x109 platelets). However, in the converse crossoverexperiments, EASs from control animals actively accumulated platelets when studied in the AV shunts of n-3FA-treated animals (1.8+0.4x 109 platelets; p<0.01 versus n-3FA-treated EASs in shunts ofnormal animals). Dietary n-3FAs also abolished vascular lesion formation at sites of carotid endarterectomy 6 weeks after surgery (cross-sectional area of neointima 0.048±0.031 mm2 compared with0.428±0.104 mm2 in control arteries; p=0.010). ConclusionIn nonhuman primates, dietary n-3FAs in high doses eliminate both vascular thrombus formation and vascular lesion formationafter mechanical vascular injury while largely sparing hemostatic function and modestly reducing blood thrombotic responses. These effects are attributed to selective n-3FA-dependent alterations in cellular membrane functions.

Enhanced In Vivo Antithrombotic Effects of Endothelial Cells Expressing Recombinant Plasminogen Activators Transduced With Retroviral Vectors

BACKGROUND The effects of regulating endothelial cell (EC) plasminogen activator production on thrombus accumulation in vivo are incompletely understood. By overexpressing plasminogen activators in ECs via gene transfer, the hypothesis was tested that increased levels of plasminogen activators inhibit the accumulation of thrombus in vivo. METHODS AND RESULTS Cultured baboon ECs transduced with human cDNAs for wild-type tissue plasminogen activator (TPA) or for glycosylphosphatidylinositol-anchored urokinase-type plasminogen activator (a-UPA) were seeded onto collagen-coated segments of vascular graft (collagen segments) and exposed overnight to flow using an in vitro perfusion circuit. The antigenic levels of TPA and UPA each increased 10-fold in the media perfusing the corresponding transduced ECs compared with untransduced ECs (P < or = .05 in both cases). In baboons the antithrombotic effects of TPA-transduced or a-UPA-transduced ECs were measured as 111In-platelet deposition and 125I-fibrin accumulation on collagen segments bearing sparsely attached ECs (tarnsduced versus untransduced) interposed in exteriorized arteriovenous femoral shunts. Platelet-rich thrombus formed on the collagen segments with fibrin-rich thrombus propagated distally. The presence of TPA-transduced or a-UPA-transduced ECs on collagen segments at a density of 25,000 ECs/cm2 decreased 111AIn-platelet deposition and 125I-fibrin accumulation on collagen surfaces compared with untransduced ECs present at equivalent density (P < .05 for platelet deposition with TPA-transduced ECs and P < .05 for platelet deposition on the propagated tail, as well as fibrin accumulation on the graft with a-UPA-transduced ECs). The systemic levels of fibrinopeptide A, thrombin-antithrombin complex, D-dimer, and both local and systemic levels of TPA and UPA were not increased by transduced ECs compared with untransduced ECs. The focal antithrombotic effects of transduced ECs appear to be due to local enhancement of thrombolysis. CONCLUSIONS ECs transduced with recombinant TPA and a-UPA enhance local antithrombotic activity in vivo. This strategy of attaching transduced ECs overexpressing plasminogen activators may be therapeutically useful by preventing thrombo-occlusive failure of implanted cardiovascular devices or mechanically denuded vessels.

The Role of Alpha and Beta Platelet-Derived Growth Factor Receptor in the Vascular Response to Injury in Nonhuman Primates

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.

Antithrombotic and Antilesion Benefits without Hemorrhagic Risks by Inhibiting Tissue Factor Pathway

The effects of inhibiting tissue factor-dependent thrombus formation on vascular neointimal lesion formation have been evaluated by inhibiting tissue factor activity using intravenous injections of active-site inactivated recombinant factor VIIa (FVIIai) administered to baboons immediately prior to initiating bilateral femoral balloon artery angioplasty and surgical carotid endarterectomy. FVIIai abolished thrombus formation at sites of vascular injury and decreased vascular lesion formation by approximately 50 percent at 30 days. We conclude that thrombus formation at sites of vascular injury is predominately tissue factor-dependent and that transient inhibition of tissue factor activity prevents both vascular thrombosis and vascular lesion formation, which implies that transiently inhibiting tissue factor at the time of elective mechanical vascular procedures may be useful in reducing clinical restenosis.

Antithrombotic effects of combining activated protein C and urokinase in nonhuman primates.

BackgroundWe have determined in vivo the relative antithrombotic efficacy and hemostatic safety of combining low-dose activated protein C (APC) and urokinase (urinary plasminogen activator, u-PA), two natural proteins that regulate thrombogenesis. Methods and ResultsTo model acute thrombotic responses of native blood under conditions of arterial flow, thrombogenic segments of Dacron vascular graft (VG) were incorporated into chronic exteriorized femoral arteriovenous (AV) access shunts in baboons. Thrombus formation on VG was determined by measuring 1) the deposition of autologous 111In platelets using real-time scintillation camera imaging, 2) the accumulation of 125I fibrin, 3) segment patency by Doppler flow analysis, and 4) blood tests for thrombosis, including plasma concentrations of platelet factor 4, 8-thromboglobulin, fibrinopeptide A (FPA), and D-dimer. Treatments consisting of low-dose and intermediate-dose APC (0.07 or 0.25 mg/kg hr), u-PA (25,000 or 50,000 IU/kg hr), or the combination were administered for 1 hour by continuous intravenous infusion. In untreated controls, platelets and fibrin accumulated rapidly, reaching plateau values at 1 hour of 15.1 % 3.8x i09 platelets and 7.8 % 2.2 mg fibrin. Although the low-dose APC or u-PA alone did not decrease either platelet or fibrin deposition significantly, this combination moderately reduced both platelet and fibrin accumulation (7.3 ± 2.6x 109 platelets, p < 0.05; 3.9 ± 0.6 mg fibrin, p < 0.05). Furthermore, intermediate-dose APC or u-PA reduced thrombus formation by half when administered alone (p < 0.001 for both platelet and fibrin deposition), and the combination markedly interrupted the accumulation of platelets (3.0 % 1.0X 109 platelets, p < 0.001) and fibrin (13 % 0.6 mg fibrin, p < 0.001). During active treatments, all VG segments remained patent. Hemostatic plug forming capability, as measured by template bleeding times, remained normal during all experiments (p > 0.05). The T;% clearance time for APC activity was not affected by the concurrent administration of u-PA. u-PA alone increased the plasma levels of D-dimer, FPA, and, interestingly, APC, implying that during pharmacological activation of the fibnrnolytic system, thrombin activity was released, and the protein C pathway was activated. ConclusionsA combination of intermediate-dose APC and u-PA produce substantial and efficient antithrombotic effects without impairing hemostatic function.

Antithrombotic Effects of Orally Active Synthetic Antagonist of Activated Factor X in Nonhuman Primates

BACKGROUND Since activated factor X (FXa) has a central role in hemostasis and thrombosis, it is an attractive target for antithrombotic strategies. Accordingly, we evaluated the relative antihemostatic and antithrombotic effects of an orally active amidinoaryl propanoic acid inhibitor of FXa, APAP, in baboons. METHODS AND RESULTS With a two-component thrombogenic device that induced the concurrent formation of both arterial-type platelet-rich and venous-type fibrin-rich thrombus when interposed in chronic exteriorized arteriovenous (AV) femoral shunts flowing at 40 mL/min, thrombus formation was compared for oral versus parenteral APAP by measurement of 111In-platelet deposition, 125I-fibrin accumulation, thrombotic obstruction of flow, and circulating levels of blood biochemical markers of thrombosis. The direct infusion of APAP (120 micrograms/min) into AV shunts proximal to thrombogenic devices for 1 hour achieved local drug levels of 4.3 +/- 0.4 mg/L and substantially reduced the accumulation of platelets and fibrin in the formation of venous-type fibrin-rich thrombus (P < .01) but not in the formation of platelet-rich arterial-type thrombus (P > .1). APAP was subsequently removed from plasma with plasma clearance rates of T50 alpha of 6.3 minutes and T50 beta of 99 minutes. The oral administration of APAP (50 mg/kg) produced peak plasma levels of 3.7 +/- 1.4 micrograms/mL at 30 minutes and gradually declining plasma levels over about 6 to 8 hours, with bioavailability estimated to be approximately 5% to 12%. Oral APAP decreased platelet deposition (P < .01) and fibrin accumulation (P < .05) in venous-type thrombus but failed to decrease platelet or fibrin accumulation in arterial-type thrombus (P > .1 in both cases). Oral and infused APAP prolonged the activated partial thromboplastin time and prevented thrombus-dependent elevations in plasma fibrinopeptide A, thrombin-antithrombin III complex, beta-thromboglobulin, and platelet factor 4 levels. Additionally, APAP produced dose-dependent inhibition of FXa bound to thrombus on segments of vascular graft interposed in exteriorized AV shunts for 15 minutes. CONCLUSIONS An oral synthetic antagonist of FXa, APAP, inhibits the formation of venous-type fibrin-rich thrombus by inactivating bound and soluble FXa without impairing platelet hemostatic function.