Abstract 450: Phosphorylation Of Platelet αIIbβ3 Is Crucial For Arterial Thrombosis In Vivo And Microparticle Generation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Manojkumar Valiyaveettil ◽  
Weiyi Feng ◽  
Ganapati Mahabaleshwar ◽  
David Phillips ◽  
Tatiana Byzova ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Annexin V ◽  
Vessel Occlusion ◽  
Occlusion Time ◽  
Fibrinogen Receptor

Functional activity of platelet fibrinogen receptor αIIbβ3 is crucial for hemostasis and thrombosis. The process of αIIbβ3 activation in platelet aggregation is tightly regulated. It has been previously shown that β3 subunit of the complex undergo tyrosine phosphorylation, which, in turn, is believed to control recruitment of several intracellular adaptors. Mutations of Tyr747/ Tyr759 within the cytoplasmic domain of αIIbβ3 (DiYF substitution) were found to result in reversible platelet aggregation. To assess whether αIIbβ3 tyrosine phosphorylation is critical for arterial thrombosis, we utilized intravital microscopy to monitor thrombus formation in vivo in WT and DiYF mice. Compared to WT, DiYF mice exhibited delayed platelet adhesion and reduced rate of thrombus formation at the initial stages of thrombosis. Likewise, isolated DiYF platelets exhibited reduced adhesion to collagen under in vitro sheer conditions compared to WT. The progression phase of thrombosis in vivo was similar in WT and DiYF mice. The most dramatic difference was observed at the final phase of thrombus formation since it took 3-times longer for blood vessels in DiYF mice to occlude compared to WT. To specifically address the role if β3 phosphorylation in platelet αIIbβ3 vs αvβ3 on leukocytes and vascular cells, we transfused labeled WT and DiYF platelets into irradiated WT mice with low blood cells and platelet counts. It was found that transfusion of DiYF but not WT platelets resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion time in vivo. Similar differences were observed in tail bleeding test. Importantly, we have found that the lack of β3 phosphorylation impaired an ability of platelets to generate microparticles in response to activation, an event believed to be critical for the final stages of thrombosis. When stimulated with thrombin and PMA, DiYF platelets shed ~50% less Annexin V-positive microparticles as compared to WT platelets. Thus, β3 tyrosine phosphorylation of αIIbβ3 in platelets is crucial for the microparticles generation by activated platelets and the overall process of arterial thrombosis in vivo.

Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3442-3442 ◽  
Author(s):  
Reheman Adili ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Reheman Adili ◽  
Katherine Mast ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
High Shear ◽  
Vessel Occlusion ◽  
12 Lipoxygenase ◽  
Induced Aggregation

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

Apolipoprotein AIV (ApoA-IV) Is a Novel Ligand of Platelet β3 Integrin That Negatively Regulates Platelet Adhesion, Aggregation, and Thrombosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 156-156
Author(s):  
Christopher M. Spring ◽  
Wuxun Jin ◽  
Hong Yang ◽  
Adili Reheman ◽  
Guangheng Zhu ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Protein S ◽  
Vessel Occlusion ◽  
Platelet Adhesion And Aggregation ◽  
Β3 Integrin

Abstract Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

Discrimination Between Prostglandin-Dependent And-Independent Functions Of Essential Fatty Acids In Arterial Throm- Bogenesis

1981 ◽  
Author(s):  
U M T Houtsmuller ◽  
G Hornstra ◽  
E Haddeman
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Arterial Thrombus ◽  
Induced Aggregation

Arterial thrombus formation is reduced in essential fatty acid (EFA) deficiency. This goes together with an enhanced thrombin induced aggregation of platelets in vitro,whereas collagen-induced aggregation is definitely suppressed. A small amount of linoleic acid (18:2 (n-6)) is able to cure EFA-deficiency and to normalize arterial thrombogenesis. This latter effect may be due to either the structural function of this EFA or to its function as the ultimate dietary precursor of prostaglandins (PG). Columbinic acid, a stereo-isomer of γ-linolenic acid (18:3 (n-6)) was recently shown to possess all the structural functions of EFA, but not the PG-dependent ones. This fatty acid therefore presents a suitable tool to investigate the PG-dependence of arterial thrombogenesis and its underlying processes. We therefore compared the effect of small amounts of linoleic and columbinic acid (both as methylesters) on the water vapour release in vivo (which is a sensitive parameter for a non-PG dependent function of polyenoic fatty acids), arterial thrombosis tendency (time needed for the thrombotic obstruction of an aorta prosthesis) and platelet aggregation in vitro (aggregometry) induced by collagen and thrombin. In contrast to linoleic acid, columbinic acid did not normalize arterial thrombosis tendency and collagen induced platelet aggregation. Columbinic acid was equally effective as linoleic acid in the normalization of the water vapour release in vivo and of the thrombin-induced aggregation. We conclude that arterial thrombus formation and collagen- induced aggregation greatly depend on prostanoid formation, whereas thrombin-induced aggregation does not. The structural role of polyenoic fatty acids in thrombin-induced aggregation may provide a tool in the elucidation of factors determining the thrombin-sensitivity of blood platelets.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

scholarly journals ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

2018 ◽  
Vol 10 (4) ◽  
pp. 44
Author(s):  
Mihir K Patel ◽  
Kiranj K. Chaudagar ◽  
Anita A. Mehta
Keyword(s):  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Platelet Activity ◽  
Aggregation Assay ◽  
Thrombosis Model ◽  
Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

C57BL/6JOlaHsd Mice with Tandem Deletion of the Multimerin 1 and Alpha-Synuclein Genes Have Impaired Platelet Function in Vivo and in Vitro That Can Be Corrected by Multimerin 1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3926-3926 ◽  
Author(s):  
Subia Tasneem ◽  
Adili Reheman ◽  
Heyu Ni ◽  
Catherine P.M. Hayward
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Knockout Mice ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Alpha Synuclein ◽  
Type I ◽  
Significant Difference

Abstract Studies of mice with genetic deficiencies have provided important insights on the functions of many proteins in thrombosis and hemostasis. Recently, a strain of mice (C57BL/6JOlaHsd, an inbred strain of C57BL/6J) has been identified to have a spontaneous, tandem deletion of the multimerin 1 and α-synuclein genes, which are also adjacent genes on human chromosome 4q22. Multimerin 1 is an adhesive protein found in platelets and endothelial cells while α-synuclein is a protein found in the brain and in blood that is implicated in neurodegenerative diseases and exocytosis. In vitro, multimerin 1 supports platelet adhesion while α-synuclein inhibits α-granule release. We postulated that the loss of multimerin 1 and α-synuclein would alter platelet function and that recombinant human multimerin 1 might correct some of these abnormalities. We compared platelet adhesion, aggregation and thrombus formation in vitro and in vivo in C57BL/6JOlaHsd and C57BL/6 mice. Thrombus formation was studied by using the ferric-chloride injured mesenteric arteriole thrombosis model under intravital microscopy. We found that platelet adhesion, aggregation and thrombus formation in C57BL/6JOlaHsd were significantly impaired in comparison to control, C57BL/6 mice. The number of single platelets, deposited 3–5 minutes after injury, was significantly decreased in C57BL/6JOlaHsd mice (P <0.05, platelets/min: C57BL/6 = 157 ± 15, n=16; C57BL/6JOlaHsd = 77 ± 13, n=17). Moreover, thrombus formation in these mice was significantly delayed. Thrombi in C57BL/6JOlaHsd were unstable and easily dissolved, which resulted in significant delays (P<0.001) in vessel occlusion (mean occlusion times: C57BL/6 = 15.6 ± 1.2 min, n=16; C57BL/6JOlaHsd = 31.9 ± 2.1 min, n=17). We further tested platelet function in these mice by ADP and thrombin induced platelet aggregation using platelet rich plasma and gel-filtered platelets, respectively. Although no significant differences were seen with ADP aggregation, thrombin-induced platelet aggregation was significantly impaired in C57BL/6JOlaHsd mice. Platelet adhesion to type I collagen (evaluated using microcapillary chambers, perfused at 1500 s−1 with whole blood) was also impaired in C57BL/6JOlaHsd mice. However, platelets from C57BL/6JOlaHsd mice showed a normal pattern of agonist-induced release of α-granule P-selectin. Multimerin 1 corrected the in vitro aggregation and adhesion defects of C57BL/6JOlaHsd platelets. Furthermore, the transfusion of multimerin 1 into C57BL/6JOlaHsd mice corrected the impaired platelet deposition and thrombus formation in vivo. No significant difference was found in tail bleeding time between the two groups of mice. As α-synuclein knockout mice have a shortened time to thrombus formation (Circulation2007;116:II_76), the effects of multimerin 1 on impaired platelet function in C57BL/6JOlaHsd mice provide supportive evidence that multimerin 1 contributes to platelet adhesion and thrombus formation at the site of vessel injury. The findings suggest multimerin 1 knockout mice will be useful to explore platelet function. The first two authors and participating laboratories contributed equally to this study.

A Locally Administered Novel Heparin Complex Attenuates Collagen-Induced Platelet Activation and Thrombosis In Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3361-3361
Author(s):  
Riitta Lassila ◽  
Annukka Jouppila ◽  
Ulla M Marzec ◽  
Stephen R Hanson
Keyword(s):  
Blood Flow ◽  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Ptfe Graft ◽  
Thrombin Time ◽  
Thrombosis Model ◽  
Baboon Model ◽  
Induced Aggregation

Abstract Abstract 3361 We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002). Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen. In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008). In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures: Lassila: Aplagon: Chief Scientific Advisor.

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