Mediates Platelet Aggregation Research Articles

Protease activated receptor-1 (PAR-1) mediated platelet aggregation is dependant on clopidogrel response

IntroductionClopidogrel inhibits ADP mediated platelet aggregation through inhibition of the P2Y12 receptor by its active metabolite. Thrombin induces platelet aggregation by binding to protease activated receptor-1 (PAR-1), and inhibition of PAR-1 has been evaluated in patients treated with clopidogrel to reduce ischemic events after acute coronary syndromes. Residual PAR-1 mediated platelet aggregation may be dependent on extent of clopidogrel response. Material and MethodsPlatelet aggregation was measured in 55 patients undergoing elective PCI at 16–24hours after 600mg clopidogrel loading dose by light transmittance aggregometry using ADP 20μM and thrombin receptor agonist peptide (TRAP) at 15μM and 25μM as agonists. Genomic DNA was genotyped for common CYP2C19 variants. ResultsIncreasing quartiles of 20μM ADP induced platelet aggregation after clopidogrel loading were associated with increasing levels of TRAP mediated platelet aggregation. Patients in the highest quartile (clopidogrel non-responders) of post treatment ADP aggregation had significantly higher TRAP mediated aggregation than the patients in the lowest quartile (clopidogrel responders) [TRAP 15μM: 79.6±5% vs. 69.5±8%, p<0.001]. ConclusionsNon-responders to clopidogrel show increased residual platelet aggregation induced by TRAP, whereas clopidogrel responders exhibit attenuated response to TRAP. Addition of PAR-1 antiplatelet drugs may be most effective in patients with reduced clopidogrel response and high residual TRAP mediated platelet aggregation.

Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

The disulfide isomerase ERp57 mediates platelet aggregation, hemostasis, and thrombosis

AbstractA close homologue to protein disulfide isomerase (PDI) called ERp57 forms disulfide bonds in glycoproteins in the endoplasmic reticulum and is expressed on the platelet surface. We generated 2 rabbit Abs to ERp57. One Ab strongly inhibited ERp57 in a functional assay and strongly inhibited platelet aggregation. There was minimal cross-reactivity of this Ab with PDI by Western blot or in the functional assay. This Ab substantially inhibited activation of the αIIbβ3 fibrinogen receptor and P-selectin expression. Furthermore, adding ERp57 to platelets potentiated aggregation. In contrast, adding a catalytically inactive ERp57 inhibited platelet aggregation. When infused into mice the inactive ERp57 prolonged the tail bleeding times. We generated 2 IgG2a mAbs that reacted with ERp57 by immunoblot. One of these Abs inhibited both ERp57 activity and platelet aggregation. The other Ab did not inhibit ERp57 activity or platelet aggregation. The inhibitory Ab inhibited activation of αIIbβ3 and P-selectin expression, prolonged tail bleeding times, and inhibited FeCl3-induced thrombosis in mice. Finally, we found that a commonly used mAb to PDI also inhibited ERp57 activity. We conclude that a glycoprotein-specific member of the PDI family, ERp57, is required for platelet aggregation, hemostasis, and thrombosis.

Chlorthalidone: The Forgotten Diuretic

Chlorthalidone's safety and efficacy in the management of hypertension has been demonstrated in landmark trials. Despite understanding the effects of thiazides on urinary sodium excretion and intravascular volume, the exact mechanism of their antihypertensive effects is not clearly understood. Common compensatory mechanisms for decreases in circulating plasma volume include increased adrenergic tone and systemic vascular resistance, as well as increases in the renin–angiotensin–aldosterone system. Chlorthalidone has been shown to decrease platelet aggregation and vascular permeability and promote angiogenesis in vitro, which is thought to be, in part, the result of reductions in carbonic anhydrase—dependent pathways, including catecholamine–mediated platelet aggregation and downregulation of VEGF–C gene expression. This article reviews the comparative clinical data between chlorthalidone and hydrochlorothiazide, the pharmacologic properties that might explain some of their differences regarding half–life and efficacy, and what is known about the effect of chlorthalidone on intermediate endpoints.

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

Abstract 3361We 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.

Comparative Studies on the HIT Antibody Mediated Platelet Aggregation / Serotonin Release by Synthetic Pentasaccharide and Two Chemoenzymatically Synthesized Heptasaccharides

Abstract 2231Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT pentasaccharide-binding sites within their structures which is comparable to the pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the pentasaccharide in heparin compromised patients. Disclosures:No relevant conflicts of interest to declare.

Unlike Heparins Newer Oral Anticoagulants Do Not Interact with HIT Antibodies and Maybe Useful in the Longterm Anticoagulant Management of Heparin Compromised Patients

Abstract 2317 Introduction:Heparin and low molecular weight heparins such as enoxaparin and dlateparin are widely used in the management of thrombosis and cardiovascular disorders. However these anticoagulants are capable of producing the generation of HIT antibodies and their use is not recommended in patients with previous history of HIT. More recently several new oral anticoagulants have been approved around the world. These include the oral anti-Xa agents namely, Apixaban (Bristol-Myers Squibb), Rivaroxaban(Bayer Healthcare) and anti-IIa agents, dabigatran (Boehringer-Ingelheim). All of these represent synthetic low molecular weight compounds. Apixaban and Rivaroxaban are active agents where as Dabigatran requires endogenous activation. The purpose of this investigation was to determine the relative effects of the newer anticoagulants and enoxaparin on HIT antibody mediated platelet aggregation and other interactions with platelets. Materials:Rivaroxaban was obtained in powdered form from Bayer Healthcare (Wuppertal, Germany) Apixaban and Dabigatran were of synthetic origin. Enoxaparin was obtained from Sanofi-Aventis (paris, France). All drugs were dissolved in appropriate solutions and a working solution of 100 ug/ml was prepared in buffered saline. Pooled HIT sera was prepared by pooling clinically symptomatic HIT patients with high titers of anti-heparin platelet factor 4 antibodies. Method:Whole blood samples drawn from normal healthy volunteers were supplemented with each of these agents at a graded concentration of 0–100 ug/ml for 60 minutes to determine the relative release of platelet factor 4. To test the interaction of HIT antibody with each of these agents 50 ul of PRP samples were mixed with 150 ul HIT positive heat inactivated sera/plasma or plasmapheresis fliud (collected from symptomatic HIT patients). Graded amounts of each of these new anticoagulants at 0.1, 1.0 and 10 ug/ml were added to test the platelet aggregation response at a period of 60 minutes. Results:In contrast to enoxaparin which produced an increase in the platelet factor 4 release upon 60 minute incubation 25.6+3.1 ng/ml), none of the new oral anticoagulants produced an increase in the PF4 <15.0 ng/ml. Enoxaparin also produced a strong HIT antibody mediated response, whereas none of these newer oral agents produced any aggregation responses. Conclusion:These studies demonstrate that –enoxaparin the newer oral anticoagulant drugs do not interact with HIT antibody to mediate plarelet aggregation responses. Moreover, these newer agents do not promote platelet factor 4 release. Thus, these agents can be used in the long term anticoagulant management of heparin compromised patients. Disclosures:No relevant conflicts of interest to declare.

TCT-167: Protease Activated Receptor-1 (PAR-1) Mediated Platelet Aggregation is Dependant on Clopidogrel Response

TCT-167: Protease Activated Receptor-1 (PAR-1) Mediated Platelet Aggregation is Dependant on Clopidogrel Response

Gene Targeting Implicates Cdc42 GTPase in GPVI and Non-GPVI Mediated Platelet Filopodia Formation, Secretion and Aggregation

BackgroundCdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation.Methodology/Principal FindingsWe utilized the Mx-cre;Cdc42lox/lox inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42+/+ mice. Platelets isolated from Cdc42−/−, as compared to Cdc42+/+, mice exhibited (a) diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b) inhibition of filopodia formation on immobilized CRP or fibrinogen, (c) inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d) inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e) minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42−/− mice compared with Cdc42+/+ mice.Conclusion/SignificanceOur data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

Fibrinogen concentrate for management of bleeding

Fibrinogen concentrate for management of bleeding

The Generation of An Aptamer Inhibitor of Murine Von Willebrand Factor (VWF) Mediated Platelet Aggregation

AbstractAbstract 4312Venous thromboembolism (VTE) is a national health concern, with an occurrence of over 900,000 cases per year and over 300,000 deaths per year. The total number of cases of VTE and the incidence of VTE-related deaths exceeds those related to both myocardial infarction and stroke. With an aging population, the incidence of VTE has also been increasing. Current treatment of venous thromboembolism with anti-coagulation is not optimal. There is a risk of bleeding, thrombus extension, pain and swelling as well as a recurrence rate of 3–9%. A significant inflammatory response occurs with venous thromboembolism. This inflammation can influence the extent of thrombosis, vein wall fibrosis and valve damage in the thrombosed vein. In a high percentage of VTE patients a condition of venous insufficiency known as post-thrombotic syndrome (PTS) can develop. PTS is associated with stasis ulceration, dermatitis and edema. Venous thrombogenesis is influenced by platelet (PLT) and leukocyte (WBC) adhesion as well as interactions between these cells. There is growing evidence to suggest that VWF interactions with PLT GPIbα can mediate some of these early events. This is evidenced by the reduction in PLT/WBC recruitment and reduced thrombus growth seen in either VWF or GPIbα deficient mice. These data point to a role for VWF in VTE. We sought to develop an aptamer to mouse VWF that would inhibit its interactions with platelet GPIbα. The availability of this tool would support investigations into the role of VWF in mouse models of VTE.Aptamers are oligonucleotides that fold into three-dimensional structures and specifically bind to ligands with high affinity. Aptamers bound to proteins can modify and/or inhibit protein-protein interactions. Using an in vitro selection method known as Systematic Evolution of Ligands by EXponential enrichment (SELEX), we generated aptamers that bind to murine VWF (mVWF) from a modified RNA pool. Nine of these aptamers bind to mVWF with single-digit or sub- nanomolar affinity. A subset of these aptamers also binds to human VWF (hVWF). The aptamers that bind to hVWF inhibit platelet adhesion/aggregation in human whole blood. Further in vitro characterization has demonstrated that five of these aptamers specifically inhibit the interaction between mVWF and recombinant human GPIbα, but do not interfere with the binding of mVWF to collagen. These five aptamers were also active in vivo in a FeCl3-induced thrombosis model in mice. Intravenous injection of the anti-mVWF aptamers prolonged time to occlusion from a baseline of 10–15 minutes to either 25–35 minutes or >40 minutes in this model, depending on the aptamer.These results demonstrate that we have identified high affinity aptamers to mVWF that specifically disrupt mVWF binding to platelets and have an antithrombotic effect in an in vivo mouse model of thrombosis. These aptamers will allow us to investigate the role of VWF in murine models of venous thrombosis. This project was supported by Award Number R01HL095091 from the National Heart, Lung, And Blood Institute. Disclosures:Woelfel:Archemix Corporation: Employment. Wagner:Archemix Corporation: Employment. McGinness:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.

Differential Inhibition of Arachidonic Acid Mediated Aggregation of Platelets by Branded and Generic Argatroban Preparations

AbstractAbstract 5131Argatroban represents a parentral antithrombin agent which is used in the management of anticoagulation in heparin compromised patients. Its main mechanism of action is mediated via inhibition of thrombin and its generation. While its effect on platelet activation inhibition by thrombin have been reported, very little information on the effects of this drug on thromboxane formation and Arachidonic Acid mediated activation of platelets is available. Argatroban and its generic versions namely Slovastan, Argaron and Gartban, may modulate Arachidonic Acid mediated platelet aggregation and release processes. To test this hypothesis a branded version of Argatroban (Mitsubishi – Tanade, Tokyo, Japan) and three generic versions of Argatroban namely Slovastan, Gartban, and Slovastan were compared for their effects on Arachidonic Acid mediated aggregation of platelets in normal healthy male and female volunteers (and n = 4). Other agonists such as Epinephrine, Collagen, and ADP were also used. The effect of Arachidonic Acid on serotonin release was also measured using an Elisa technique for the measurement of serotonin. All of the generic and branded versions of Argatroban produce varying levels of the inhibition of the Arachidonic Acid mediated aggregation of platelets, ranging from the 24 to 36 percent in comparison to the control at a concentration of 1 mg/ mL (p value < 0.05). Interestingly all of the Argatrobans produced a relatively weaker inhibition of the Arachidonic Acid mediated aggregation of platelets 24–26 percent inhibition versus 36 percent at the final concentration of 1 mg/mL. No differences were noted in the aggregation profile of ADP, Collagen, and Epinephrine between the control and the Argatrboban at a final concentration of 1 mg /mL. No differences were noted between the generic and branded Argatroban on all of the other agonists induced aggregation. In the serotonin release assays, all of the generic and branded Argatroban produced a concentration dependent inhibition of serotonin release which was stronger with the branded version of Argatroban. These results indicate that besides the inhibition of thrombin Argatroban is capable of inhibiting platelet activation via other mechanisms. Moreover, the generic versions of Argatroban exhibit a weaker inhibition of Arachidonic Acid mediated platelet aggregation and release. These studies suggest that beside thrombin mediated aggregation Argatroban and its generic versions can modulate platelet activation and release reactions. Furthermore a difference is observed between the generic and branded product which may impact the safety inefficacy profile in these agents. Argatroban is used commonly in the management of patients with heparin induced thrombocytopenia where multiple mechanisms of platelet activation are contributory to the pathogenesis of this syndrome. Modulation of thrombanxane formation and platelet release mechanism by Argatroban may represent an additional mechanism of the clinical effects of this parentral antithrombin agent. Disclosures:No relevant conflicts of interest to declare.

Role of DNAJC10, An Hsp40 Type Chaperone, In αIIbβ3 Biogenesis In Human Megakaryocytes

AbstractAbstract 2111 INTRODUCTION:The integrin αIIbβ3 mediates platelet aggregation by binding to fibrinogen and other plasma proteins. Inherited defects of αIIbβ3 result in the mucocutaneous bleeding disorder, Glanzmann thrombasthenia (GT). However, pharmacological inhibition of αIIbβ3 can reduce mortality after myocardial infarction and percutaneous stent placement. Thus the degree to which αIIbβ3 functions is critical. One element influencing the degree of αIIbβ3 function is its level of surface expression on the platelet. The post-translational mechanisms controlling surface expression remain unclear, but their stringency is evident in certain GT patients whose full length αIIbβ3 complexes are completely retained and degraded intracellularly. A clearer understanding of the post-translational processes controlling αIIbβ3 surface expression can provide insight into a pathway that is a potential target for novel αIIbβ3-targeted therapies. Toward that end we previously reported the interaction of αIIb with DNAJC10, an hsp40 type chaperone protein, which was identified by a proteomic analysis of αIIb-interacting proteins. Continuing that investigation, we have explored the function of DNAJC10 in αIIbβ3 biogenesis pathway. METHODS:Megakaryocytes were derived from human umbilical cord blood (UCB) by enrichment of UCB leukocytes for CD34+ progenitor cells followed by culture in serum-free medium with thrombopoietin and stem cell factor. Under our conditions the UCB leukocytes differentiate into a population of large cells of which > 90% express αIIbβ3, > 80% express GPIb, and about 50% express α2β1. Interaction of αIIb and β3 with DNAJC10 was tested by co-immunoprecipitation with mAbs specific for αIIb (CA3, M148, B1B5), b3 (7H2), or the αIIbβ3 complex (10E5). Megakaryocytes were treated with a proteasome inhibitor (MG-132) to increase the capture of αIIb (and potential interacting proteins) that had been routed to the proteasome for degradation. DNAJC10, αIIb, and β3 localization within megakaryocytes and platelets was observed with immunofluorescence, and compared to localization of the endoplasmic reticulum (ER) and Golgi. The effect of siRNA-mediated knockdown of DNAJC10 on αIIbβ3 expression was assessed by flow cytometry and immunofluorescence. RNA depletion was confirmed by quantitative RT-PCR. RESULTS:DNAJC10 co-immunoprecipitated with αIIb-specific (CA3, M148, B1B5), β3-specific (7H2), and complex-specific (10E5) mAbs, indicating that it associated with αIIbβ3 in megakaryocytes. An antibody (B1B5) that recognized pro-αIIb and pro-αIIbβ3 co-immunoprecipitated much more DNAJC10 than antibodies preferentially recognizing mature αIIb and αIIbβ3. The amount of DNAJC10 immunoprecipitated was markedly increased after cells were treated with a proteasome inhibitor. siRNA against DNAJC10 resulted in significantly elevation of αIIbβ3 surface expression on megakaryocytes as judged by flow cytometry. Immunofluorescence with confocal microscopy surprisingly revealed that DNAJC10, nominally an endoplasmic reticulum protein, did not colocalize with ER markers, but was distributed in foci throughout the megakaryocytes, proplatelet processes, platelet buds, and mature peripheral blood platelets. CONCLUSIONS:DNAJC10 engages the precursor forms of αIIb and αIIbβ3 at a point upstream of degradation and may be an intermediate step in targeting these to the proteasome. Since siRNA mediated knockdown of DNAJC10 resulted in increased αIIbβ3 surface expression, this chaperone may be a negative regulator of αIIbβ3 surface expression. Disclosures:No relevant conflicts of interest to declare.

Structural and Functional Analysis of the C-Terminus of Gαq in Complex with the Human Thromboxane A2 Receptor Provides Evidence of Constitutive Activity

The human thromboxane A(2) (TXA(2)) receptor (TP) is known to mediate platelet aggregation and vasoconstriction. The receptor predominantly interacts with the Gq protein, thereby activating phospholipase C and increasing the intracellular calcium level. In this study, we synthesized a 15-residue peptide corresponding to the C-terminal domain of the Gq protein alpha subunit (Galphaq-Ct peptide) and characterized its interaction with recombinant TP purified from a baculovirus expression system in the presence and absence of an agonist using fluorescence and NMR spectroscopic studies. With fluorescence binding assays, we demonstrated that the Galphaq-Ct peptide was bound to TP, in the absence of the agonist, with a K(d) value of approximately 17 muM. Interestingly, upon addition of the agonist, U46619, the Galphaq-Ct peptide's binding affinity for this activated TP was reduced, thereby increasing the K(d) value to approximately 240 muM. NMR experiments demonstrated that the TP-bound Galphaq-Ct peptide shows a different affinity and conformation, in the absence and presence of the agonist, U46619. This suggested there is the possibility of ligand-free constitutive TP signaling through Galpha binding. Thus, an HEK293 cell line that stably expresses human TP and lacks the ability to produce TXA(2) was created by gene transfer and G418 selection. In comparison with the control cells, the stable cell line showed significant Galpha-mediated ligand-free calcium signaling. The study indicates a promising new outlook for the examination of prostanoid receptor-G-protein interactions in greater detail using integrated NMR spectroscopy, the purified receptor, and the stable cell line.

The protective effects of paclitaxel on platelet aggregation through the inhibition of thromboxane A2 synthase

Paclitaxel is an anticancer drug used in the treatment of ovarian, breast, head and neck, lung, and prostate cancer. We investigated the antiplatelet activity of paclitaxel in vitro as well as a possible antiplatelet mechanism. Paclitaxel inhibited washed rabbit platelet aggregation induced by collagen in a concentration dependent manner, with an IC(50) of 59.7 +/- 3.5. However, it had little effect on platelet aggregation mediated by arachidonic acid, U46619, a thromboxane (TX) A(2) mimic, or thrombin, suggesting that paclitaxel may strongly inhibit collagen mediated signal transduction. In accordance with these findings, paclitaxel blocked collagen induced cytosolic calcium mobilization, arachidonic acid liberation, and serotonin secretion. In addition, it inhibited arachidonic acid mediated platelet aggregation by about 37% by interfering with TXA(2) synthase as measured by the formation of arachidonic acid mediated TXA(2) and prostaglandin D(2), as well as cyclooxygenase-1 and TXA(2) synthase activity assays. Taken together, these results point to a cellular mechanism for the antiplatelet activity of paclitaxel through the inhibition of TXA(2) synthase and cytosolic calcium mobilization. This may contribute to the beneficial effects of paclitaxel on the cardiovascular system.

Metabolism and Disposition of the Thienopyridine Antiplatelet Drugs Ticlopidine, Clopidogrel, and Prasugrel in Humans

Ticlopidine, clopidogrel, and prasugrel are thienopyridine prodrugs that inhibit adenosine-5'-diphosphate (ADP)-mediated platelet aggregation in vivo. These compounds are converted to thiol-containing active metabolites through a corresponding thiolactone. The 3 compounds differ in their metabolic pathways to their active metabolites in humans. Whereas ticlopidine and clopidogrel are metabolized to their thiolactones in the liver by cytochromes P450, prasugrel proceeds to its thiolactone following hydrolysis by carboxylesterase 2 during absorption, and a portion of prasugrel's active metabolite is also formed by intestinal CYP3A. Both ticlopidine and clopidogrel are subject to major competing metabolic pathways to inactive metabolites. Thus, varying efficiencies in the formation of active metabolites affect observed effects on the onset of action and extent of inhibition of platelet aggregation (IPA). Knowledge of the CYP-dependent formation of ticlopidine and clopidogrel thiolactones helps explain some of the observed drug-drug interactions with these molecules and, more important, the role of CYP2C19 genetic polymorphism on the pharmacokinetics of and pharmacodynamic response to clopidogrel. The lack of drug interaction potential and the absence of CYP2C19 genetic effect result in a predictable response to thienopyridine antiplatelet therapy with prasugrel. Current literature shows that greater ADP-mediated IPA is associated with significantly better clinical outcomes for patients with acute coronary syndrome.

The GPIIbIIIa antagonist drugs eptifibatide and tirofiban do not induce activation of apoptosis executioner caspase-3 in resting platelets but inhibit caspase-3 activation in platelets stimulated with thrombin or calcium ionophore A23187

Platelet surface receptor, glycoprotein (GP) IIbIIIa (integrin αIIbβ3), mediates platelet aggregation and plays a key role in hemostasis and thrombosis.[1][1],[2][2] Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation.[3][3] Two of these antagonists

Different Characteristics of Direct Factor Xa Inhibitors: In Vitro Comparative Studies of Rivaroxaban and Apixaban.

Abstract 4170 IntroductionRivaroxaban and apixaban are selective, reversible but structurally different direct Factor Xa inhibitors that are in late-stage clinical development for the prevention and treatment of venous and arterial thrombosis. Studies in animal models demonstrated antithrombotic efficacy with these new agents (Perzborn E et al. J Thromb Haemost 2005;3:514–521; Wong PC et al. J Thromb Haemost 2008;6:820–829). The objective of this study was to characterize and compare in vitro rivaroxaban and apixaban in functional assays. MethodsFactor Xa activity, rate constants (kon/koff), and prothrombinase activity were measured using purified Factor Xa and measuring the cleavage of chromogenic (Factor Xa), fluorogenic substrates (kon/koff), or using prothrombin as a substrate in a reconstituted prothrombinase complex and measuring the activity of the generated thrombin in the presence of a thrombin-specific chromogenic substrate. Clotting times and thrombin generation (TG) were measured using commercially available kits. Tissue factor (TF)-mediated platelet aggregation was measured in defibrinated plasma. ResultsRivaroxaban and apixaban showed similar affinity for free Factor Xa (Ki 0.4 and 0.6 nM, respectively), comparable association (kon 1.7 × 107 M–1 s–1 and 0.88 × 107 M–1 s–1, respectively) and dissociation (koff 5 × 10–3 s–1 and 2.4 × 10–3 s–1, respectively) rates, and inhibition of prothrombinase-bound Factor Xa (2.1 nM and 2.7 nM, respectively; Table). However, in human plasma-based systems, the 2 agents showed different potency. Despite the reported comparable plasma protein binding for apixaban and rivaroxaban (87% and 92–95%, respectively), higher concentrations of apixaban were needed to inhibit TG and TF-mediated platelet aggregation, and to prolong clotting time, compared with rivaroxaban. The concentrations needed to double clotting times, such as prothrombin time, activated partial thromboplastin time, clotting times in thromboelastometric measurements triggered either by the extrinsic (ex-TEM®) or intrinsic (in-TEM®) coagulation pathway, and prothrombinase-induced clotting time, were 3- to 8-fold higher for apixaban than for rivaroxaban (Table). The IC50 values for reducing the peak TG and endogenous thrombin potential in the TG assay were 3-fold higher for apixaban (0.20 and 4.96 μM, respectively) compared with rivaroxaban (0.06 and 1.48 μM, respectively; Table). The IC50 values for inhibiting TF-mediated platelet aggregation were 8-fold higher for apixaban (0.51 μM) versus rivaroxaban (0.06 μM; Table). ConclusionsThese results demonstrate that structurally different Factor Xa inhibitors may differ in their antihemostatic potency in spite of comparable affinity to Factor Xa, and suggest that rivaroxaban may be a more potent anticoagulant and indirect inhibitor of platelet aggregation than apixaban.TableCharacteristics of rivaroxaban and apixaban in in vitro studiesRivaroxabanApixabanDifference in potencyAffinity constant (Ki)0.4 nM0.6 nMRate constants (kon, koff)1.7 × 107 M–1 s–1, 5 × 10–3 s–10.88 × 107 M–1 s–1, 2.4 × 10–3 s–1Prothrombinase (IC50)2.1 nM2.7 nMThrombin generation (5 pM tissue factor)Peak thrombin generation (IC50)0.06 μM0.20 μM3-foldEndogenous thrombin potential (IC50)1.48 μM4.96 μM3-foldCoagulation assays (concentrations needed to double clotting times)Prothrombin time0.69 μM5.43 μM8-foldActivated partial thromboplastin time2.07 μM7.14 μM3-foldProthrombinase-induced clotting time0.17 μM0.68 μM4-foldThrombelastography (ex-TEM®)0.34 μM1.46 μM4-foldThrombelastography (in-TEM®)0.87 μM6.22 μM7-foldTissue factor-induced platelet aggregation (IC50)0.06 μM0.51 μM8-fold Disclosures:Perzborn:Bayer Schering Pharma AG: Employment. Tersteegen:Bayer Schering Pharma AG: Employment. Harwardt:Bayer Schering Pharma AG: Employment. Lange:Bayer Schering Pharma AG: Employment.

The GPIIbIIIa Antagonist Drugs Eptifibatide and Tirofiban Inhibit Caspase-3 Activation in Thrombin- and Calcium Ionophore-Stimulated Human Platelets.

Abstract 2998Poster Board II-976 Introduction:The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods:The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion:We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P>0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P<0.01, n=7). However, when platelets were preincubated with eptifibatide and tirofiban before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation by an average of 44-50% (P<0.05, n=7). The fact that eptifibatide and tirofiban do not promote caspase-3 activation in unstimulated platelets suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. The inhibitory effect of eptifibatide and tirofiban on thrombin- and A23187-induced caspase-3 activation suggests a role of GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. Conclusions:We have demonstrated a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures:No relevant conflicts of interest to declare.

Oversulfated Chondroitin Sulfate Does Not Cause Augmentation in HIT Antibody Mediated Heparin-Induced Platelet Aggregation (HIPA).

Abstract 2417Poster Board II-394Heparin is one of the widely used anticoagulants for the prophylaxis and treatment of thrombotic disorders. Heparin, a linear polysaccharide belonging to a family of glycosaminoglycans (GAGS) consists of repeating disaccharide units of 1-4 linked iduronic acid and glucosamine residues. Heparin is a mixture of low, medium and high molecular fractions and is heterogeneous not only in terms of molecular weight but also sites and degree of sulfation. Oversulfated chondroitin sulfate, one of the important toxic contaminants in certain batches of heparins manufactured during 2007 to 2008 resulted in deaths of some patients following intravenous administration that led to the recall of these heparins by the FDA. An increased incidence of heparin-induced thrombocytopenia (HIT) and thrombosis syndrome (HITTS) was reported as a result of contaminated heparins. It was hypothesized that OSCS interacts with heparins and other LMWHs increasing their HIT/HITTS potential. In order to validate this hypothesis blood was drawn from healthy volunteers (n=5) and mixed with 3.2% sodium citrate solution (9 parts of whole blood to 1 part of citrate). The citrated blood samples were centrifuged at 800 rpm to obtain platelet rich plasma (PRP) and further centrifuged at 3000 rpm to obtain platelet poor plasma (PPP). Apheresis fluid was collected from confirmed cases of HIT and pooled. The platelet aggregometers (Biodata) were blanked with individual PPP. The PRP (300ml) was mixed with HIT positive pooled apheresis fluid (200ml) and platelet aggregation was recorded using Gentium Heparin, Enoxaparin, and Bemiparin at a final concentration of 10mg/ml, either alone or in mixtures of 10%, 20% and 30% OSCS. The Heparin-induced platelet aggregations (HIPA) were run for a period of 30 minutes and the tracings obtained. While OSCS alone at a final concentration of 10 mg/ml caused an average of 29.5% platelet aggregation, it did not cause an augmentation of platelet aggregation in 10%, 20% and 30% mixtures with Heparin, Enoxaparin, Bemiparin and AVE 5026 (see table). This data suggests that drugs like heparin, and low molecular weight heparins such as Enoxaparin, and Bemiparin did not augment HIT/HITTS response when combined in various proportions with OSCS. Based on HIPA the reported increase in HIT response may therefore be due to in vivo interactions of the OSCS with platelets and endothelial cells. Corroborative evidence from clinical studies are warranted to validate these preliminary results.Drugs (FC10mg/ml)Drug Alone10%OSCS mixture20%OSCS mixture30%OSCS mixtureOSCS aloneSalineHeparin52.3%60%58.3%58.6%21.6%8.3%Enoxaparin56%58%60.5%60.5%31%7%Bemiparin60%59.6%63%60%37.6%9.3% Disclosures:No relevant conflicts of interest to declare.