Carotid Artery Thrombosis Model Research Articles

COX‐1+/−COX‐2−/− genotype in mice is associated with shortened time to carotid artery occlusion through increased PAI‐1

We found a high incidence of thrombotic deaths in COX-1(+/-)COX-2(-/-) mice and sought to define the mechanism of these events. The cyclooxygenase products thromboxane A(2) and prostacyclin are important in the regulation of coagulation but their role in fibrinolysis is largely unexplored. PAI-1 blocks fibrinolysis by inhibiting plasminogen activator. Our objective was to explain the mechanism of increased thrombosis associated with the COX-1(+/-)COX-2(-/-) genotype. Carotid artery occlusion times were measured after photochemical injury. PAI-1 levels were measured in the plasma by ELISA. PAI-1 levels in the aorta were measured by RT-PCR and Western blotting. Urinary metabolites of Thromboxane A(2) and prostacyclin were measured by ELISA. The COX-1(+/-)COX-2(-/-) genotype is associated with a decreased time to occlusion in the carotid artery thrombosis model (30 ± 5 minutes vs 60 ± minutes in wild type, p<.001). The COX-1(-/-)COX-2(+/+), COX-1(+/-)COX-2(+/-) and COX-1(+/-)COX-2(+/+) all had occlusion times similar to wild type. COX-1(+/+)COX-2(-/-) had a prolonged occlusion time. COX-1(+/-)COX-2(-/-) had increased PAI-1 levels in the plasma and aorta and with a prolonged euglobulin lysis time (37.4 ± 10.2 hours vs 15.6 ± 9.8 hours in wild type, p<.004). The decreased time to occlusion in the COX-1(+/-)COX2(-/-) mice was normalized by an inhibitory antibody to PAI-1 whereas the antibody had no effect on the time to occlusion in wild type mice. The COX-1(+/-)COX-2(-/-) genotype is associated with a shortened time to occlusion in the carotid thrombosis model and the shortened time to occlusion is mediated through increased PAI-1 levels resulting in decreased fibrinolysis.

Acute Pulmonary Embolism (PE)—Is Echocardiography Underutilised in Regional Australia

Results: Although slope and offset of the baroreflex peripheral arc (linear relation) were similar, the neural arc response (inverted sigmoid curve) range was attenuated in SHR (71± 2% vs. 91± 3%, P< 0.01). The operating-point AP was higher in SHR than in WKY (144± 6mmHg vs. 109± 5mmHg, P< 0.01). Although NE was higher in SHR at baseline condition (403.1 pg/ml vs. 203.2 pg/ml, P< 0.01), AP response to phenylephrine was similar (linear relation), suggesting preserved peripheral response to sympathetic activity. Conclusion: The baroreflex equilibrium diagram indicates that baroreflex regulation of SNA rather than the cardiovascular response to SNA plays a critical role in the its non-targeted control (CD39 fused to a non-functional mutated single-chain antibody). Most importantly in a mouse model of ferric chloride-induced carotid artery thrombosis, targeted-CD39 was protective against vessel occlusion at a concentration at which the non-targetedCD39was ineffective (p< 0.005).At the same concentration no tail bleeding prolongation was observed for the targeted-CD39 while the ineffective non-targeted-CD39 showed a bleeding tendency (p< 0.01). Conclusion: Targeting CD39 to its desired site of action enables administration of such a low concentration as to avoid the previously observed bleeding tendencies while still being a highly effective antithrombotic drug. Thus, enriching CD39 to activated platelets at growing thrombi prevents the previously limiting bleeding side effects and advances CD39 towards potential clinical use. doi:10.1016/j.hlc.2011.05.024

Prolylcarboxypeptidase Deficiency Is a Risk Factor for Arterial Thrombosis and Hypertension

AbstractAbstract 651 Background.Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angiotensin II, and alpha melanocyte stimulating hormone and activates prekallikrein to plasma kallikrein (Blood 103:4554, 2004). In GWAS, it has been recognized as a risk factor for metabolic syndrome, hypertension, and pre-eclampsia. We postulated that PRCP murine hypomorphs (PRCPgt/gt) have a cardiovascular phenotype. Methods and Results.PRCP is mostly found in kidney in proximal tubules. In arteries, it is found both on endothelium and in media. A gene-trap murine hypomorph was created with 7% mRNA and 23% PRCP antigen in renal tissue. Using the Rose Bengal carotid artery thrombosis models, PRCPgt/gt mice had shorter carotid artery occlusion times (24±3 min [mean±SD]) compared to wild type (52±8 min). On a 4% ferric chloride carotid artery thrombosis assay PRCPgt/gt occluded in 21±8 min whereas wild type do not occlude at 60 min. Pharmacologic inhibition of PRCP with Z-pro-prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-Chloromethylketone or PKSI-572 in 3 mouse strains also shortened the time to carotid artery occlusion. PRCPgt/gt were constitutively hypertensive during the late night cycle (122±5 mm Hg mean arterial pressure vs 114±6 mm Hg for wildtype) as measured by carotid artery telemetry. Treatment of these animals with the mitochondria specific antioxidant mitoTEMPO significantly reduced (113±7 mm Hg) the elevated BP. Plasma angiotensin II and bradykinin levels were unaltered in PRCPgt/gt. PRCPgt/gt plasma had a significant increase in contact activation-induced thrombin generation. Aortic and renal reactive oxygen species (ROS) were increased (3.2-fold and 2.8-fold, respectively) in PRCPgt/gt mice as determined by dihydroethidium (DHE) fluorescence. PRCPgt/gt aortic and renal superoxide measured by lucigenin luminescence also was increased (1.6-fold and 1.7-fold, respectively). In PRCPgt/gt kidneys Amplex Red fluorescence, a measure of hydrogen peroxide, was increased 2.4-fold. Renal tissue had 1.6-fold increased uncoupled eNOS on SDS-PAGE. Arterial occlusion times in PRCPgt/gt were corrected by treatment with antioxidant apocynin or tempol. PRCP siRNA knockdowns in HUVEC or mesenchymal embryonic fibroblasts prepared from PRCPgt/gt embryos had increased constitutive DHE fluorescent ROS (2.1-fold and 1.4-fold, respectively). PRCPgt/gt aorta had decreased expression of Kruppel-like factors 2 and 4, thrombomodulin and eNOS. Moreover, PRCP knockdowns in HUVEC had 36% reduced eNOS mRNA expression. Conclusion.These investigations indicate that PRCP is a specific gene/protein target for arterial thrombosis risk and hypertension. Its presence modulates constitutive cell and tissue ROS. Arterial thrombosis risk is related to effect of ROS on endothelial cell anticoagulant mechanisms. Disclosures:No relevant conflicts of interest to declare.

Biphasic Roles for the Soluble Guanylyl Cyclase (sGC) In Platelet Activation In Mice

AbstractAbstract 486The role of intracellular secondary messenger cGMP in platelet activation has been controversial, with both stimulatory and inhibitory roles reported. The platelet cGMP is believed to be predominantly synthesized by soluble guanylyl cyclase (sGC), which is activated by nitric oxide (NO). To specifically determine the role of sGC-dependent cGMP synthesis in platelet function and in vivo thrombosis and hemostasis, we produced mice harboring a “floxed” sGC beta1 allele. In the “floxed” sGC beta1 mice (sGC beta1fl/fl), the exons 7 and 8 of sGC beta1 gene and an inserted Neo cassette were flanked with three LoxP sites. Platelet-specific deletion of sGC beta1fl/fl allele was accomplished through breeding of the sGC beta1fl/fl mice with pf4-Cre recombinase transgenic mice. Immunoblotting showed the complete absence of this protein in sGC beta1fl/fl/Cre platelets. Mice lacking sGC beta1 in platelets appeared to develop normally and had normal blood counts, including platelets. Blood pressure of platelet-specific sGC deficient mice was comparable to that of wild-type littermates. Inactivating the sGC beta1 gene in platelets abolished cGMP production induced by either NO donors or platelet agonists that are known to activate endogenous NO synthesis, confirming that both the platelet agonist-induced and NO donor-induced platelet cGMP production are predominantly mediated by sGC. Platelets lacking sGC exhibit a marked defect in aggregation and secretion in response to low doses of platelet agonists, collagen and thrombin. Importantly, tail-bleeding times were significantly prolonged in the platelet-specific sGC deficient mice compared with the wild-type littermates. In a FeCl3-induced carotid artery thrombosis model, time to occlusive thrombosis was prolonged in the platelet-specific sGC deficient mice, compared to wild type littermates. Thus, the agonist-stimulated sGC activation is important in promoting platelet granule secretion and aggregation. On the other hand, NO donor SNP-induced inhibition of platelet activation was abolished in sGC-deficient platelets. However, at high concentrations (>100μM), SNP inhibited platelet activation in both wild type and sGC deficient mice, indicating that both cGMP-dependent and -independent mechanisms are involved in NO donor-induced inhibition of platelet activation. Together, our data demonstrate that sGC contributes to both agonist-induced platelet activation and NO donor-induced platelet inhibition. Disclosures:No relevant conflicts of interest to declare.

M-AAA-Thrombin: Potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1

Background Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus, several antithrombotic agents targeting thrombin have been developed. We previously reported on a thrombin derivative prepared by dual chemical modifications designated as 'M-anhydrothrombin', which possessed both anticoagulant and antiplatelet properties. In order to obtain a more potent antithrombotic thrombin derivative, we prepared a recombinant thrombin mutant and its chemically-modified derivative, and examined their antithrombotic efficacies. Methods and Results We prepared a thrombin mutant, 65A43A205A-Th (designated as “AAA-Th”) in which Lys65(70) 1 1 Numbers in bracket are those of chymotrypsinogen numbering system. , and His43(57) and Ser205(195) were replaced by Ala, and its chemically-modified derivative at the carboxyl groups (designated as "M-AAA-Th"). M-AAA-Th possessed no enzymatic activity, but retained high affinity and specificity for factor VIII, and prolonged the APTT with a slight effect on PT and no effect on TT. Platelet aggregation induced by PAR1 activation was also suppressed by M-AAA-Th. In contrast, conventional thrombin inhibitors, argatroban and hirulog, substantially prolonged the TT rather than the APTT and the PT. In thromboelastgraph assays, M-AAA-Th suppressed whole blood clotting in a dose-dependent manner, and its effect was synergistically enhanced in the presence of soluble thrombomodulin (s-TM). M-AAA-Th also demonstrated a potent antithrombotic property in the FeCl 3-induced carotid arterial thrombosis model in guinea pigs with minimum effects on the APTT and PT and no prolongation of the TT. Conclusion M-AAA-Th may be a potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.

Effect of Chinese herbal medicine for activating blood circulation and detoxifying on expression of inflammatory reaction and tissue damage related factors in experimental carotid artery thrombosis rats

To observe the pharmaceutical effect of Chinese drugs for activating blood circulation (Xiongshao Capsule, XSC, ) and for activating blood circulation and detoxification (Xiongshao Capsule and Huanglian Capsule, XSHLC, ) in terms of the indices of thrombosis, inflammatory reaction and tissue damage related factors in experimental carotid artery thrombosis rats. Fifty Wistar rats were randomly divided into the sham operation group, the model group, the Simvastatin group (SG), the activating blood circulation (ABC) group, and the activating blood circulation and detoxifying (ABCD) group, with 10 rats in each group. Simvastatin (1.8 mg/kg), XSC (0.135 g/kg) and XSHLC (0.135 g/kg) were administered to Simvastatin, ABC and ABCD group by gastrogavage, and an equal volume of normal saline was given to the sham operation group and the model group. After 2 weeks of successive medication, the rats in the model and all drug therapy groups were made into experimental carotid artery thrombosis model. The serum levels of matrix metalloproteinases (MMP-9), tissue inhibitors to metalloproteinase (TIMP-1), granule membrane protein-140 (GMP-140), tissue-type plasminogen activator (t-PA), high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6) were detected with enzyme-linked immunoassay 24 h later. Compared with the model group, the levels of serum GMP-140, hs-CRP, IL-6 and MMP-9 were significantly decreased, and the level of t-PA was significantly increased in the ABC and ABCD group ( P<0.05), while the level of serum hs-CRP in ABCD group decreased significantly compared with that in the ABC group (P<0.05). Chinese drugs both for activating blood circulation and for activating blood circulation and detoxifying have good effects on regulating indices of thrombosis, inflammatory reaction and tissue damage in experimental carotid artery thrombosis rats. The effect of activating blood circulation and detoxifying drugs on regulating the level of serum hs-CRP is superior to that of activating blood circulation drug alone.

Fibrolase: Trials and Tribulations

Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the β-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.

Drug Rediscovery: Glybenclamide Exhibits Thromboxane Receptor Dependent Antithrombotic Activity

Occlusive disorders such as myocardial infarction and ischemic stroke require platelet activation, a central axis being the synthesis and binding of thromboxane A2 (TXA2) to its receptor (abbreviated as TPR). There are currently no TPR antagonists available for clinical use and there are profound obstacles in translating novel formulations into a therapeutic. One solution is to investigate new uses for currently prescribed drugs, an approach termed “drug rediscovery”. To that aim, it was noticed that the sulfonylurea, glybenclamide, appears to share several pharmacophores with the TPR antagonist SQ29,548. Thus, we predicted it would interact with TPRs. It was found that glybenclamide: 1) inhibited human platelet aggregation induced by the TPR agonist U46619 (1 uM) and the TXA2 precursor arachidonic acid (0.5 mM); 2) concentration‐dependently (1–10 uM) displaced SQ29,548; 3) failed to inhibit aggregation stimulated by 15 uM ADP, or the thrombin receptor activating‐peptide 4 (40 uM); 4) failed to raise cAMP levels; 5) selectively (10mg/kg) blocked mouse TPR‐mediated aggregation ex vivo; 6) prolonged the tail bleeding time in mice; and 7) prolonged the time for occlusion in a mouse carotid artery thrombosis model. These findings indicate that glybenclamide can exert an inhibitory effect on platelet function by specifically interacting with TPR and thus has potential in the management of thrombotic disorders.

A Novel Inhibitor of Plasminogen Activator Inhibitor-1 Provides Antithrombotic Benefits Devoid of Bleeding Effect in Nonhuman Primates

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterial venous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latter's therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time.

Biological effects of fucoidan isolated from Fucus vesiculosus on thrombosis and vascular cells

BackgroundFucoidan is a highly sulfated glycosaminoglycan, which has a molecular structure similar to that of heparin. The antithrombotic effects of fucoidan in vitro have been widely reported, but its antithrombotic effects in vivo as well as its other biological properties in vitro have not been well investigated.MethodsThis study investigated the effects and mechanism of fucoidan from Fucus vesiculosus on thrombosis both in vitro and in vivo. A ferric chloride-induced mouse carotid artery thrombosis model was used to determine the antithrombotic effects of fucoidan in vivo. Additionally, changes in the levels of proinflammatory cytokines and chemokines were examined in vascular cells treated with fucoidan.ResultsIn vivo studies employing a ferric chloride-induced mouse carotid artery thrombosis model indicated that fucoidan had a stronger antithrombotic activity than heparin. Further, vascular cells treated with fucoidan demonstrated a decrease in proinflammatory cytokine and chemokine production as well as inhibition of proliferation.ConclusionThe major findings of this study showed that fucoidan has a stronger antithrombotic effect than heparin in vivo and that fucoidan has an inhibitory effect on proinflammatory cytokine production and proliferation of vascular cells.

A Rice Variety (HYJA-Ri4) Enhances Arterial Thrombus Formation in an Animal Model of Thrombosis

Epidemiological evidence exists for a causative role of inappropriate diets in the development and clinical outcome of thrombotic diseases. Our previous studies have shown that some fruits and vegetables have antithrombotic activity. As rice is a principal ingredient of everyday food in Japan, the aim of the present study was to determine rice varieties with antithrombotic effect. Western-style high-fat diet containing flour prepared from whole-grain rice of different varieties was fed to atherosclerosis-prone (apolipoprotein E- and low density lipoprotein receptor-deficient) mice for 3 months. A laser-induced carotid-artery thrombosis model was used. None of the five tested rice varieties showed an antithrombotic effect. The effect of HYJA-Ri1, HYJA-Ri2, HYJA-Ri3 and HYJA-Ri5 rice on experimental thrombus formation did not differ significantly from the controls. However, HYJA-Ri4 rice significantly enhanced the thrombotic reaction (p = 0.0078), thus having a prothrombotic effect. Our findings suggest that HYJA-Ri4 rice should not be included in an antithrombotic diet.

Drug Rediscovery: Glybenclamide Exhibits Thromboxane Receptor-Dependent Antithrombotic Activity.

Abstract 2994Poster Board II-971While proper platelet function is a vital component of hemostasis, inappropriate activation of platelets contributes to occlusive disorders such as myocardial infarction, and stroke. One pathway for platelet activation involves the synthesis of the lipid mediator thromboxane A2 (TXA2), which acts by binding to its seven-transmembrane receptor (abbreviated as TPR). Although this signaling pathway participates in the genesis of thrombosis, currently, there are no TPR antagonists that are available for clinical use; and aspirin remains the sole agent targeting this pathway. However, since aspirin is associated with multiple adverse effects, there is still considerable interest in developing TPR antagonists. To this end, we took the “drug rediscovery” approach to identify TPR antagonists. Since the sulfonylurea glybenclamide appeared to possess most of the pharmacophores known to exist in the TPR antagonist SQ29,548, we predicted it would interact with TPRs (and exhibit antiplatelet/antithrombotic activity). It was found that glybenclamide: 1) inhibited human platelet aggregation induced by the TPR agonist U46619 (1μM) and the TXA2 precursor arachidonic acid (0.5mM), concentration-dependently (1-10μM); 2) displaced SQ29,548 from its binding sites; 3) lacked any detectable effects on aggregation stimulated by 15μM ADP, or the thrombin receptor activating-peptide 4 (40μM); 4) failed to raise cAMP levels; 5) selectively (at 10mg/kg) blocked mouse TPR-mediated aggregation, under ex vivo settings; 6) prolonged (at 10mg/kg) the tail bleeding time in mice; and 6) prolonged (at 10mg/kg) the time for occlusion in a mouse carotid artery thrombosis model. Taken together, these findings indicate that glybenclamide exerts inhibitory effects on platelet function by interacting with TPR, and exhibits antithrombotic activity. Thus glybenclamide, or a rationally designed derivative, has the potential to be applied in the management of thrombotic disorders. Disclosures:No relevant conflicts of interest to declare.

Investigation of the Anti-Platelet Activity of a Novel Function-Blocking Antibody Targeting the Human Thromboxane A2 Receptor Protein.

Abstract 4019Poster Board III-955Despite the well-documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally designed antagonists available for clinical use. To a large extent this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. To this end, while previous work has defined the C-terminal segment of the second extracellular loop (C-EL2) of TPR as the ligand binding site, we have recently mapped the amino acid residues, within this region, which mediate ligand interaction. Since these studies employed a polyclonal antibody targeting the EL2 region of the receptor protein, and since EL2 has been shown to contain the ligand binding domain, we hypothesized that this antibody (referred to hereafter as C-EL2Ab) would exhibit biological activity. On this basis, the purpose of the current study was to investigate the capacity of C-EL2Ab to block TPR-mediated function, using human and mouse platelets. Our initial results demonstrated the C-EL2Ab blocked human and mouse platelet aggregation triggered by 1μM of the TPR agonist U46619, or 0.5mM of the thromboxane A2 mimetic arachidonic acid, in a dose-dependent fashion (150-250nM). On the other hand, control experiments revealed that C-EL2Ab, even at 250 nM, did not produce any detectable effects on platelet activation by ADP (15μM) or the thrombin receptor activating-peptide 4 (40μM). Together, these findings suggest that C-EL2Ab acts as a selective antagonist for TPRs. Consistent with this notion, we found that C-EL2Ab has the ability to displace a radio-labeled TPR antagonist (i.e., [3H]SQ29,548) from its TPR binding sites, both in human and mouse platelets. We next examined the anti-platelet activity of C-EL2Ab under ex vivo experimental settings. It was found that intravenous (IV) tail injections of 250nM of C-EL2Ab resulted in significant blockade of platelet aggregation by 1μM U46619, but not by 15μM ADP. Finally, using a mouse carotid artery thrombosis model, our data demonstrated that C-EL2Ab (at 250 nM; administered IV) prolonged the time for occlusion. Thus, C-EL2Ab has the potential to protect against thrombosis development; which is consistent with the established link for TPRs in thrombotic disorders. In summary, these studies show that C-EL2Ab dose-dependently blocks platelet aggregation induced by the TPR pathway, under both in vitro and ex vivo conditions, and that these effects are mediated via direct interaction with the receptor protein. Furthermore, IV injections of C-EL2Ab are found to confer protection against thrombosis development, in mice. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, mediated via its binding to platelet TPRs, making it the first function-blocking antibody against these receptors. Moreover, the identification of a functionally active TPR sequence will significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity. Disclosures:No relevant conflicts of interest to declare.

Abstract 5304: Ectonucleoside Triphosphate Diphosphohydrolase-1 (ENTDP-1/CD39) Over-Expression Delays in vivo Carotid Arterial Thrombosis in Mice

Background: Vascular injury results in release adenosine diphosphate (ADP), activating platelets leading to further release of ADP from platelet granules. ADP release results in platelet aggregation and cross-linking. Ectonucleoside Triphosphate Diphosphohydrolase-1 (ENTDP-1/ CD39), an endothelial cell-expressed diphosphonucleotidase, rapidly converts ATP and ADP to AMP which is further degraded by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic, vasodilatory and cardioprotective compound. We hypothesized that transgenic CD39 over-expression would increase the time to thrombosis in an in vivo ferric chloride-induced carotid artery thrombosis model. Methods: Mice, 8 –12 week old human CD39 overexpressing (hCD39-OE) and wildtype littermates, were anesthetized using 55 mg/kg ketamine and 15 mg/kg xylazine, intubated, and ventilated with room air using a mouse respirator (Harvard Apparatus). A midline incision was made and the left carotid artery was exposed. Baseline carotid blood flow was obtained using a Doppler flow probe (Model 0.5 VB, Transonics Systems). Thrombosis was induced by placing filter paper saturated with 10% FeCl3 on the carotid artery for 3 minutes. Carotid blood flow was monitored continuously and time to cessation of flow analyzed. Results: In response to FeCl3 induced injury, wild-type mice demonstrated a consistent time to occlusion of 8±1 minute. In contrast, in animals overexpressing hCD39, the time to occlusion following FeCl3 induced injury was profoundly increased to 60±2 minutes (&gt;7-fold). (Figure ). Conclusion: CD39 over-expression significantly delays the time to arterial thrombosis.

AβPP/APLP2 Family of Kunitz Serine Proteinase Inhibitors Regulate Cerebral Thrombosis

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Enhanced thrombosis and platelet aggregation in mice lacking alpha(1,3)‐fucosyltransferase‐IV and ‐VII are P‐selectin independent

P‐selectin (P‐sel) is a leukocyte adhesion molecule with prothrombotic functions. Fucosylation of its ligand, PSGL‐1, by alpha(1,3)‐fucosyltransferases (FucT)‐IV or ‐VII is required for binding activity. FucT‐IV(‐/‐)/VII(‐/‐) mice have an unexpected prothrombotic phenotype. We used a photochemical‐induced injury model of carotid artery thrombosis and whole blood platelet aggregation to test whether the prothrombotic phenotype was dependent on P‐sel/PSGL‐1 binding interactions. The time to occlusive thrombosis was decreased in FucT‐IV(‐/‐)/VII(‐/‐) mice [19.5±3.0 min, n=10, vs WT=34.0±2.8 min, n=12; p=0.05]. In contrast, the time to occlusive thrombosis was not altered in PSGL‐1(‐/‐) mice [25.7±2.9 min, n=6, vs WT=23.1±1.3 min, n=7; p&gt;0.05], or in Psel(‐/‐) mice [63.0±8.6 min, n=4, vs WT=45.5±7.8 min, n=4; p&gt;0.05]. ADP‐stimulated whole blood platelet aggregation in FucT‐IV(‐/‐)/VII(‐/‐) mice had a shorter lag time [22.2±0.5 sec, n=8 vs WT=27.0±0.6 sec, n=8; p=0.05] and a faster rate [21.4±0.9 sec−1, n=8 vs WT=16.4±1.4 sec−1, n=8; p=0.05]. In contrast, platelet aggregation in PSGL‐1(‐/‐) mice did not have an altered lag time [28.5±2.2 sec, n=8 vs WT=26.0±1.2 sec, n=16; p&gt;0.05], or rate [13.4±1.4 sec−1, n=8 vs WT=17.4±1.4 sec−1, n=16; p&gt;0.05]. The prothrombotic phenotype in FucT‐deficient mice is independent of P‐sel/PSGL‐1 binding activity. Supported by AHA FTF Award 0275023N and RO1‐HL090823.

Antithrombotic Potential of GW813893: A Novel, Orally Active, Active-site Directed Factor Xa Inhibitor

Factor Xa (FXa) has been a target of considerable interest for drug development efforts aimed at suppressing thrombosis. In this report, a new orally active, small molecule, active-site directed FXa inhibitor, GW813893, has been profiled in a succession of in vitro and in vivo assays involved in its preclinical characterization as a potential antithrombotic therapeutic. In vitro profiling of GW813893 consisted of assessing its inhibitory potential against FXa and a broad panel of related and unrelated enzymes and receptors. Additionally, the FXa inhibition potential of GW813893 was assessed in prothrombinase and plasma-based clotting assays. In vivo characterization of GW813893 consisted of thrombosis studies in a rat inferior vena cava model, a rat carotid artery thrombosis model, and a rabbit jugular thrombosis model. Bleeding studies were conducted in a rat tail transection model. Ex vivo determinations of compound effects on FX and clotting activity were also undertaken. GW813893 was more than 90-fold selective over all enzymes tested, and it inhibited FXa and prothrombinase activity with a Ki of 4.0 nM and 9.7 nM, respectively. In vivo, GW813893 concentration-dependently suppressed thrombotic activity in all models tested. The antithrombotic activity correlated with the suppression of plasma-based clotting activity and the inhibition of plasma FX activity (P < 0.02). Over the antithrombotic dose-range, an increased bleeding diathesis was not observed. These experiments demonstrate that GW813893 is a potent, selective, orally active inhibitor of FXa. The data suggest that GW813893 has robust antithrombotic potential at doses that have no detectable hemostasis liability. Collectively, the profile suggests that GW813893 has the preclinical pharmacology underpinnings of an oral antithrombotic therapeutic.

A Specific CD36-Dependent Signaling Pathway Is Required for Platelet Activation by Oxidized Low-Density Lipoprotein

Platelet hyperactivity associated with hyperlipidemia may contribute to development of a prothrombotic state. We previously showed that oxidized low-density lipoprotein (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis activated platelets in a CD36-dependent manner. We now show that mitogen-activated protein kinase c-Jun N-terminal kinase (JNK)2 and its upstream activator MKK4 were phosphorylated in platelets exposed to oxLDL. Using apoE(-/-) mice as a model of hyperlipidemia, we showed that JNK was constitutively phosphorylated in platelets in a CD36-dependent manner. Inhibition of src kinase activity reduced JNK phosphorylation by oxLDL. Immunoprecipitations revealed that active phosphorylated forms of src kinases Fyn and Lyn were recruited to CD36 in platelets exposed to oxLDL. Pharmacological inhibition of the mitogen-activated protein kinase JNK or src family kinases abolished platelet activation by oxLDL in vitro. Using a murine carotid artery thrombosis model we demonstrated CD36-dependent phosphorylation of platelet JNK within thrombi. Furthermore, pharmacological inhibition of JNK prolonged thrombosis times in wild-type but not cd36-null mice in vivo. These findings suggest that a specific CD36-dependent signaling pathway is required for platelet activation by oxLDL and may provide insights related to development of novel antiplatelet therapies more relevant to atherothrombosis than to normal hemostasis.

Disodium Disuccinate Astaxanthin Prevents Carotid Artery Rethrombosis and ex vivo Platelet Activation

Background/Aims:The disodium disuccinate derivative of astaxanthin (DDA) is a carotenoid antioxidant under development for the treatment of ischemic cardiovascular events. Recent evidence suggests that reactive oxygen species (ROS) play an important role in platelet activation. This study seeks to investigate the effects of a reactive oxygen species quencher, DDA, in a canine model of carotid artery thrombosis. Methods:After formation of an occlusive carotid thrombus, dogs were administered recombinant tissue plasminogen activator intra-arterially to achieve thrombolysis in the presence of either 0.9% NaCl solution or DDA (10–50 mg/kg i.v. infusion). Ex vivo platelet aggregation and tongue bleeding times were measured before and after drug administration. Residual thrombus mass was analyzed at the end of each experiment. Results:The data indicated a dose- dependent reduction in the incidence of carotid artery rethrombosis. In addition, platelet aggregation and thrombus weights were dose-dependently inhibited by DDA. No change was recorded in tongue bleeding time among the treatment groups. Conclusions:The data demonstrate that at the doses used in this study, DDA significantly reduced the incidence of secondary thrombosis while maintaining normal hemostasis. The results suggest that upon further study, DDA may one day find utility in revascularization procedures.

Lipopolysaccharide augments venous and arterial thrombosis in the mouse

Background Animal models of diseases are essential for therapeutic target validation, drug discovery and development. Increasing evidence has demonstrated the importance of inflammation in thrombosis. Here, murine models of vena cava thrombosis and carotid arterial thrombosis augmented by lipopolysaccharide (LPS) were established and characterized to study the association between inflammation and thrombosis. Materials and methods Murine (C57BL/6 mice) models of ferric chloride (FeCl 3)-induced carotid arterial and vena cava thrombosis were established. Thrombus formation was measured indirectly by Doppler blood flow (i.e., clot functional interference with blood flow) in the arterial thrombosis model and directly by protein content of the clot in the venous thrombosis model. An optimal concentration of FeCl 3 was defined to induce thrombus formation and used to study the effects of LPS (i.e., a well-known inflammatory stimulus under these conditions). Real-time polymerase chain reaction (PCR) was used to examine the effect of LPS on TNFα and IL-1β mRNA expression in thrombus formation. Results Dose-dependent analysis demonstrated that 2 mg/kg, i.p., LPS provided a maximal prothrombotic effect in 2.5% ferric chloride-induced vena cava thrombosis, with a 60% increase in thrombus size (n = 8, p < 0.05) compared to vehicle treatment. In contrast, 2 mg/kg LPS had no significant effect on thrombus formation in a more severe, 3.5% FeCl 3-induced vena cava thrombosis. A similar prothrombotic effect was observed for LPS in 2.5% FeCl 3-induced carotid arterial thrombosis model. Treatment of 2 mg/kg LPS significantly augmented arterial thrombosis immediately (between 5–30 minutes) following FeCl 3 injury as assessed by change of Doppler blood flow (n = 8, p < 0.05). Real-time PCR demonstrated significant induction of TNFα and IL-1β mRNA expression in the thrombus formation in the vessels in response to LPS challenge. Conclusion These data demonstrate that LPS augments thrombus formation in acute vascular injury and that LPS-augmented thrombosis might be a useful tool to study the relationship between inflammation and thrombosis.