Xinkang Wang

Effects of factor IX or factor XI deficiency on ferric chloride‐induced carotid artery occlusion in mice

Summary.  Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl3)‐induced arterial injury model in factor‐deficient mice. Various concentrations of FeCl3 were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl3. In contrast, FXI‐ and FIX‐deficient mice were fully protected from occlusion induced by 5% FeCl3, and were partially protected against the effect of 7.5% FeCl3. The protective effect was comparable to very high doses of heparin (1000 units kg−1) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail‐bleeding‐time assay. FXI‐deficient and wild‐type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8‐fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.

Effects of factor XI deficiency on ferric chloride‐induced vena cava thrombosis in mice

Summary.  Background: Increased plasma levels of coagulation factor (F) XI are a risk factor for venous thrombosis. Objective: To further explore the relationship between FXI and venous thrombosis, we evaluated FXI‐deficient and wild‐type mice in a ferric chloride (FeCl3)‐induced vena cava thrombosis model. Methods and Results: Thrombosis was induced by 3‐min topical application of filter papers containing increasing concentrations of FeCl3 and the thrombus was measured at 30 min. In contrast to wild‐type mice, FXI‐deficient mice failed to form a thrombus with 5% FeCl3, and were partially protected against 7.5% and 10% FeCl3, respectively. The protective effect was substantially stronger than a high dose of heparin (1000 units kg−1, i.v.), clopidogrel (30 mg kg−1, p.o.) or argatroban (30 mg kg−1, i.p.). These antithrombotic agents resulted in off‐scale bleeding in a tail bleeding time assay, whereas the bleeding time of FXI‐deficient mice was unchanged compared to wild‐type mice. In addition to its known effect on the coagulation cascade, enhanced clot lysis was demonstrated in FXI‐deficient mouse and human plasma compared to those supplemented with FXIa. Conclusion: Given the strong antithrombotic efficacy (possibly contributed by strong anticoagulant activity associated with increased fibrinolytic activity) and mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable therapeutic strategy to treat or prevent venous thrombosis.

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

Summary.  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 FeCl3 in C57BL/6 mice. A relatively mild stimulus (3.5% FeCl3) 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% FeCl3‐induced model without exogenous tissue plasminogen activator administration. In contrast, PCI had no effect on 3.5% FeCl3‐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.

Recombinant adenoviral expression of dominant negative IκBα protects brain from cerebral ischemic injury

Abstract Transcription factor NF-κB is associated with inflammatory response and cell survival. Under inactive condition, NF-κB is sequestered in the cytoplasm by an anchor protein, inhibitor of NF-κB (IκB). NF-κB was shown to be activated during ischemic brain injury. In the present study we have investigated the role of NF-κB in ischemic brain injury using a recombinant adenovirus expressing a dominant negative form of IκB (Adv/IκBdn) to specifically inhibit NF-κB activation. Our data demonstrated that cortical injection of Adv/IκBdn significantly reduced ischemic brain injury following permanent occlusion of the middle cerebral artery (MCAO) in rats, showing 55% reduction ( p n =8) in total ischemic lesion or 80% reduction ( p p n =8). These data provide further evidence for the role of NF-κB/IκB in ischemic brain injury and suggest that inhibition of NF-κB is neuroprotective in focal stroke.

Inhibition of Factor Xa Reduces Ischemic Brain Damage After Thromboembolic Stroke in Rats

Background and Purpose— Factor Xa (FXa) is a key coagulation protease and target for novel antithrombotic agents for prevention and treatment of diverse thromboembolic disorders. In the present study we describe the effect of a novel, potent, and selective FXa inhibitor, DPC602, on brain damage and neurobehavioral consequence in a rat thromboembolic model of stroke. Methods— Thromboembolic stroke was induced in rats by placement of an autologous clot into the middle cerebral artery. Results— Laser-Doppler monitoring of cerebral blood flow demonstrated that DPC602 (8 mg/kg, single IV/IP bolus pretreatment) markedly improved cerebral blood flow after thromboembolic stroke by 25% to 160% (n=6;P <0.001) at 1 to 6 hours. DPC602 demonstrated concentration- and time-dependent reductions in infarct size, with maximal effect (89% reduction; n=14;P <0.001) at the highest dose over controls. Neurological function was also significantly improved in DPC602-treated rats at days 1, 3, and 7 (n=13;P <0.01). DPC602 treatment did not cause cerebral hemorrhage, assessed by free hemoglobin in the ischemic brain tissues. Conclusions— These data suggest that anticoagulation with a selective FXa inhibitor might ameliorate the extent of ischemic brain damage and neurological deficits after a thromboembolic event. Enhanced clot dissolution and early reperfusion may account for the cerebrovascular-protective effect of the drug.

Comparative Analysis of Various Platelet Glycoprotein IIb/IIIa Antagonists on Shear-Induced Platelet Activation and Adhesion

Platelet accretion into arterial thrombus in stenotic arterial vessels involves shear-induced platelet activation and adhesion. The Cone and Plate(let) Analyzer (CPA) is designed to simulate such conditions in vitro under a rotating high shear rate in whole blood. In the present study, we evaluated various experimental conditions (including aspirin, temperature, and calcium concentration) and investigated the effects of small molecules along with peptide glycoprotein IIb/IIIa antagonists on platelet adhesion using the CPA system. Concentration-dependent effect of glycoprotein IIb/IIIa antagonists on shear-induced platelet adhesion showed marked differences in potencies: IC50 = 34, 35, 91, 438, and 606 nM for DPC802 (a specific glycoprotein IIb/IIIa antagonist), roxifiban, sibrafiban, lotrafiban, and orbofiban (free acid forms), respectively, and IC50 values of 43, 430, and 5781 nM for abciximab, tirofiban, and eptifibatide, respectively. Parallel study was also conducted to evaluate the effect of glycoprotein IIb/IIIa inhibitors using optical aggregometry. The potency of fibans in blocking shear-induced platelet adhesion correlated well with their binding affinity to the resting and activated glycoprotein IIb/IIIa receptors, as well as their “off-rates”. Nevertheless, none of these fibans was able to effectively block shear-induced platelet adhesion at targeted clinical dosing regimens except for abciximab. These data suggest that glycoprotein IIb/IIIa antagonists that show similar efficacy in the inhibition of platelet aggregation in a static in vitro assay may differ substantially in a shear-based system of platelet adhesion. The clinical significance of this phenomenon awaits further investigation.

Effects of orally active glycoprotein IIb/IIIa antagonists on platelet CD40 ligand (CD154) expression and platelet-heteroaggregate formation

Although intravenous treatment with glycoprotein IIb/ IIIa (GP IIb/IIIa) inhibitors is well established as beneficial in treatment of acute coronary syndromes [1], large-scale, randomized trials of oral GP IIb/IIIa inhibitors indicate an increase in mortality [2]. Several possible mechanisms for this increase have been proposed, including increased platelet aggregation at trough levels of drug [3,4], increased platelet– leukocyte heteroaggregate formation [5], CD40 ligand (CD40L)-mediated inflammatory responses [6], and other non-prothrombic toxic mechanisms [2]. In the present work, we chose to examine the effects of the GP IIb/IIIa antagonists, roxifiban and orbofiban, on two of these possibilities, increase in platelet CD40L expression and formation of platelet-heteroaggregates. There has been great interest in the potential inflammatory role of CD40L on the surface of activated platelets [7]. Platelets presenting CD40L on their surface induce endothelial cells to express tissue factor and inflammatory cytokines, suggesting a link between platelet activation and vascular wall inflammation [7,8]. Recently, oral GP IIb/IIIa antagonists have been suggested to increase the levels of platelet CD40L, thereby promoting inflammatory processes that could influence the outcome of treatment [6,9]. Another possibility to explain complications due to oral GP IIb/IIIa inhibitors is that some of these drugs may actually increase the number of platelet– leukocyte aggregates as they decrease platelet–platelet aggregates. Li et al. [10] observed that the number of platelet– leukocyte aggregates in collagen-treated blood increases in the presence of the GP IIb/IIIa inhibitor SR121566. Such aggregates could increase atherosclerotic plaque inflammation, leading to plaque destabilization, and hence, a thrombic event [5]. Roxifiban and orbofiban are both non-peptide orally active RGD mimetic compounds that block binding of fibrinogen to GP IIb/IIIa [11,12]. Roxifiban can be grouped with GP IIb/IIIa antagonists, which have relatively higher affinity to platelets and a slower off rate [13], designated Class I, while orbofiban fits into Class II, those compounds having relatively lower affinity and faster dissociation [14]. We examined the effects of both compounds on CD40L expression and platelet-heteroaggregate formation.