Target For Antithrombotic Therapy Research Articles

Thrombosis-inflammation: a novel target for antithrombotic therapy in coronary heart disease

Thrombosis-inflammation: a novel target for antithrombotic therapy in coronary heart disease

IMPACT OF CONTACT SYSTEM ON CARDIOVASCULAR DISEASES

Contact and intrinsic coagulation pathway components are involved in pathological thrombus formation. In addi­tion to coagulation, contact system activation is involved in anti/profibrinolytic and inflammatory processes that can contribute to the atherosclerotic and thrombotic en­vironment, leading to cardiovascular diseases. This lite­rature review concerns the role of the contact system on cardiovascular diseases and accentuates the potential of coagulation factors FXII (FXII) and FXI (FXI) as targets for antithrombotic therapy.

Modulation of endothelial organelle size as an antithrombotic strategy

BackgroundIt is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell‐specific secretory granules, Weibel‐Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5‐5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. ObjectiveWe hypothesized that WPB‐shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. MethodsUsing a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. Results and ConclusionsA novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro‐hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS‐CoV‐2 pandemic, with a considerable fraction of critically ill COVID‐19 patients affected by hypercoagulability, these WPB size‐reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.

Updated Understanding of Platelets in Thrombosis and Hemostasis: The Roles of Integrin PSI Domains and their Potential as Therapeutic Targets.

Platelets are small blood cells known primarily for their ability to adhere and aggregate at injured vessels to arrest bleeding. However, when triggered under pathological conditions, the same adaptive mechanism of platelet adhesion and aggregation may cause thrombosis, a primary cause of heart attack and stroke. Over recent decades, research has made considerable progress in uncovering the intricate and dynamic interactions that regulate these processes. Integrins are heterodimeric cell surface receptors expressed on all metazoan cells that facilitate cell adhesion, movement, and signaling, to drive biological and pathological processes such as thrombosis and hemostasis. Recently, our group discovered that the plexin-semaphorin-integrin (PSI) domains of the integrin β subunits exert endogenous thiol isomerase activity derived from their two highly conserved CXXC active site motifs. Given the importance of redox reactions in integrin activation and its location in the knee region, this PSI domain activity may be critically involved in facilitating the interconversions between integrin conformations. Our monoclonal antibodies against the β3 PSI domain inhibited its thiol isomerase activity and proportionally attenuated fibrinogen binding and platelet aggregation. Notably, these antibodies inhibited thrombosis without significantly impairing hemostasis or causing platelet clearance. In this review, we will update mechanisms of thrombosis and hemostasis, including platelet versatilities and immune-mediated thrombocytopenia, discuss critical contributions of the newly discovered PSI domain thiol isomerase activity, and its potential as a novel target for anti-thrombotic therapies and beyond.

Acetylcholine Inhibits Platelet Activation.

Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but fewer endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO), have been identified. Acetylcholinesterase inhibitors, such as donepezil, can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets. We measured the effect of acetylcholine or analogs of acetylcholine on human platelet activation ex vivo. Acetylcholine and analogs of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and glycoprotein IIb IIIa conformational changes. Conversely, we found that antagonists of the acetylcholine receptor, such as pancuronium, enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase, such as donepezil, also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Our data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for antithrombotic therapies.

Targeting β3 Integrin Psi Domain Inhibits Both Platelet Aggregation and Blood Coagulation: Two Birds with One Stone

Integrin αIIbβ3 plays key roles in thrombosis and hemostasis primarily through mediating platelet adhesion and aggregation. We recently reported that the active site of thiol-isomerase enzymes, CXXC motif, is expressed twice within the plexin-semaphorin-integrin (PSI) domain across all integrins and species, and the PSI domain of β3 integrin possesses endogenous thiol-isomerase activity, which may be a novel target for anti-thrombotic therapy (Blood, 2017).We developed four mouse anti-mouse β3 integrin PSI domain monoclonal antibodies (mAbs). These mAbs cross-react with β3 PSI domains of human, mouse, pig, rat, and rabbit tested but not other regions of β3 integrin, other integrins or other thiol-isomerase enzymes. They inhibit the thiol-isomerase activity of β3 PSI domain, decrease platelet adhesion/aggregation and thrombosis without increasing bleeding. Interestingly, the inhibitory effect of these mAbs on thrombosis in vivo (no anti-coagulant) was 10-20 times greater than their inhibitory effect on platelet aggregation in anti-coagulated platelet-rich plasma in vitro. This motivated us to explore whether this PSI domain contributes to blood coagulation.To asses blood clot formation and retraction, blood was incubated in non-stick tubes for two hours at 37°C in clot retraction assays. These assays showed less clot retraction and significantly lower dry clot weight in human and mouse whole blood treated with these anti-PSI mAbs compared to controls (p<0.05). As a visual representation, laser scanning confocal microscopy of platelet-rich plasma samples with labelled fibrinogen/fibrin showed that fibrin network formation of anti-PSI mAb treated clots was decreased. To measure blood coagulation in a global assay in the presence of platelets and natural blood constituents, thromboelastography (TEG) was performed. Blinded TEG showed that anti-PSI mAb-treated whole blood (human and mouse) and platelet rich plasma (human) decreased in coagulation compared to controls (p<0.05). We further compared our anti-PSI mAbs with other anti-β3 integrin mAbs that inhibit αIIbβ3 ligand binding and platelet aggregation much stronger than the anti-PSI mAbs. In this TEG assay we found that the anti-PSI mAbs were still significantly decreasing platelet coagulation parameters, much more than other inhibitory anti-β3 mAbs including M1 and JAN D1 as well as the Abciximab precursor 7E3 (p<0.05). This suggests the decreased coagulation may result from the anti-thiol-isomerase activity of the anti-PSI mAbs.Disulfide bond exchanges are required for blood coagulation and thiol-isomerase enzymes play important roles in thrombosis. Given that blood coagulation is rapidly amplified on the platelet surface (i.e. cell-based coagulation), many coagulation factors are in close proximity to β3 integrin PSI domains (with thiol-isomerase activity). The study of interaction between β3 PSI domain and coagulation factors is currently being exploring.This is the first evidence of an allosteric therapeutic target of β3 integrin. This work suggests that a novel interaction exists between platelets and the coagulation cascade that requires the β3 integrin PSI domain and its thiol-isomerase activity. Importantly, the PSI domain can be targeted to partially block both platelet aggregation and coagulation, resulting in less bleeding while still providing a strong overall anti-thrombotic effect. DisclosuresNo relevant conflicts of interest to declare.

CLEC-2 induced signalling in blood platelets

Platelet activating receptor CLEC-2 has been identified on platelet surface a decade ago. The only confirmed endogenous CLEC-2 agonist is podoplanin. Podoplanin is a transmembrane protein expressed by lymphatic endothelial cells, reticular fibroblastic cells in lymph nodes, kidney podocytes and by cells of certain tumors. CLEC-2 and podoplanin are involved in the processes of embryonic development (blood-lymph vessel separation and angiogenesis), maintaining of vascular integrity of small vessels during inflammation and prevention of blood-lymphatic mixing in high endothelial venules. However, CLEC-2 and podoplanin are contributing to tumor methastasis progression, Salmonella sepsis, deep-vein thrombosis. CLEC-2 signalling cascade includes tyrosine-kinases (Syk, SFK, Btk) as well as adapter LAT and phospholipase Cg2, which induces calcium signalling. CLEC-2, podoplanin and proteins, participating in CLEC-2 signalling cascade, are perspective targets for antithrombotic therapy.

The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy

AbstractIntegrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

Advances of Studies on Platelet GPVI as Antithrombotic Target -Review

Platelet activation is a crucial step in both physiological hemostasis and pathological thrombosis, which is an important mean to prevent and treat thrombotic diseases by inhibition of platelet activation. The current clinical antithrombotic therapy showed a high efficiency, but at risk of bleeding. Platelet glycoprotein VI (GPVI) is a platelet-specific receptor and its binding with collagen is critical for platelet activation. GPVI antagonists were shown to effectively inhibit thrombosis and inflammation without influence on normal hemostasis. As a novel target for antithrombotic therapy, it ideally combines efficacy with safety. This review summarizes the recent advances of studies on GPVI structure, function and its role in hemostasis, thrombosis, and anti-GPVI agents. The potential clinical strategies of antiplatelet drugs targeting GPVI are discussed so as to provide a reliable regimen for thrombotic diseases.

Novel pharmacological inhibitors demonstrate the role of the tyrosine kinase Pyk2 in adhesion and aggregation of human platelets.

Pyk2 is a Ca2+-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca2+-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation.

Abstract 385: Factor XII Knockout or Knockdown in Rat Produces Robust Antithrombotic Efficacy With No Bleeding

Coagulation Factor XII (FXII) is an attractive novel target for antithrombotic therapies as studies with FXIIa inhibitors and FXII knockout mouse suggest FXII is critically involved in pathological thrombus formation but is dispensable for hemostasis. To further assess the role of FXII in thrombosis and hemostasis, FXII knockout or knockdown models in rat were established and characterized. FXII knockout in rat was generated via the Zinc Finger nuclease (ZFN) technology and confirmed by lack of detectable zymogen in circulation. Titratable FXII knockdown in rat was achieved with a siRNA reagent that is potent on FXII mRNA in liver and selective against all other coagulation factors. FXII knockout in rats produced marked antithrombotic efficacy in both the arteriovenous shunt model (~100% clot weight reduction), and the 10% FeCl 3 -induced carotid artery thrombosis model (no reduction in blood flow post injury), without any increase in the cuticle bleeding time compared to the WT control rats. Ex vivo aPTT and ellagic acid-triggered thrombin generation assay (TGA) exhibited corresponding changes whereas ex vivo PT or TF (20 pM)-triggered TGA was indistinguishable from WT rats. Rats that received a single dose of FXII siRNA (at 0 (non-targeting siRNA control), or 0.01, or 0.03, or 0.1, or 0.3 mg/kg) exhibited dose-dependent knockdown in FXII mRNA in the liver and FXII zymogen in plasma (90% and 97%, respectively, for the 0.3 mg/kg dose) at day 7 post dosing. FXII knockdown was associated with dose-dependent antithrombotic efficacy (with the 0.3 mg/kg dose delivering near maximal efficacy in both models) and minimal increase in cuticle bleeding times (&lt;50% across all doses). Ex vivo pharmacodynamic markers (aPTT and TGA) tracked with the knockdown levels and efficacy. In summary, our findings in both the knockout model and the knockdown model in rats confirmed and extended literature observations with regard to the highly desirable benefit-to-risk profile of targeting FXII in preclinical species; the siRNA tool will allow detailed studies on level of target engagement needed for translatable efficacy; the KO rat will be valuable for addressing any mechanism-based safety concerns as well as off-target effects of putative FXIIa inhibitors.

RASA3 is a critical inhibitor of RAP1-dependent platelet activation.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Animal Models of Arterial and Venous Thrombosis

The intersection of the availability of genetically altered mice, new technologies for intravital microscopy and high speed computing systems has led to the use of animal models to build on the concepts that have emerged from in vitro studies of the molecular and cellular biology of hemostasis and thrombosis. In an effort to improve the understanding of the etiology and pathogenesis of thrombosis, thrombus formation has been initiated in experimental systems via mechanical disruption, laser-induced, photochemical-induced and ferric chloride-induced injury to the vessel wall, among others. None of these methods are physiologic, and as such, remain models that require extrapolation from a living animal – a mouse – to human biology. We have focused on laser-induced injury of the arteriole vessel wall in the cremasteric muscle of the mouse. Using high speed digital imaging of fluorescently labeled components and real time intravital microscopy, our group has been able to demonstrate that platelet accumulation and fibrin generation during thrombus formation occur simultaneously, that tissue factor and collagen are independent initiators of platelet activation, and that monocyte-derived microparticles deliver tissue factor to the site of thrombus development. Perhaps the most important and unanticipated observation has been that thiol isomerases, thought only to be involved in protein biosynthesis via the formation of disulfide bonds in the endoplasmic reticulum, play a critical role in thrombus formation. Protein disulfide isomerase (PDI), ERp5 and ERp57 are among the vascular thiol isomerases that are known to be important for the initiation of thrombus formation. Laser-induced thrombosis in mice is associated with PDI, ERp5 and ERp57 secretion by platelets and endothelium. Inhibition of these thiol isomerases blocks platelet thrombus formation and fibrin generation. The integrins αIIbβ3 and αVβ3 play a key role in this process, binding directly with the secreted thiol isomerases and capturing them in the vicinity of vessel wall injury. These enzymes are required for the initiation of platelet thrombus formation and fibrin generation, but the mechanism by which they function remains to be elucidated. At present, it would appear that there is an electron transfer pathway involving these enzymes that regulates the initiation of thrombus formation. The mechanism by which PDI participates in thrombus generation is being evaluated by using trapping mutant forms to identify substrates of PDI that participate in the network pathways linking thiol isomerases, platelet receptor activation and fibrin generation. Several proteins, including vitronectin, thrombospondin and Factor V, have been identified as forming covalent disulfide intermediates with PDI. We are currently exploring PDI as an antithrombotic target using isoquercetin and quercetin 3-rutinoside, inhibitors of PDI identified by high throughput screening. Tail bleeding times are equivalent for mice treated with quercetin-3-rutinoside and isoquercetin compared to untreated mice. In an in vivo mouse pulmonary embolism model, PDI inhibitors rescue a high percentage of mice from death. The b-domain of PDI binds to quercetin-3-rutinoside, and infusion of the isolated b’ domain into a mouse treated with quercetin-3-rutinoside restores thrombus formation. This suggests a method for reversal of bleeding if these PDI inhibitors are found to be complicated by bleeding. The antithrombotic properties of quercetin and isoquercetin in humans have been tested. A pharmacokinetic study with quercetin and isoquercetin determined optimal oral delivery with isoquercetin. The effectiveness of these PDI inhibitors in human studies is being evaluated in a clinical trial evaluating prophylaxis of thromboembolic events in patients with cancer-associated thrombosis. PDI is a novel target for antithrombotic therapy and is unique in that its inhibition simultaneously blocks platelet thrombus formation and fibrin generation. Disclosures Zwicker: Quercegen Pharma: Research Funding.

Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

▪The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. DisclosuresNo relevant conflicts of interest to declare.

Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis.

BackgroundInvasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear.Principal FindingsUsing Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (KD∼10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis.ConclusionsBlockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.

Abstract 54: PSI Domain of ß3 Integrin Has Endogenous Thiol Isomerase Function and is a Novel Antithrombotic Therapeutic Target

Integrin αIIbβ3 plays a critical role in platelet aggregation and adhesion, key events in hemostasis and thrombosis. Integrin activation involves complex signalling events that lead to conformational changes exposing ligand-binding sites; however, mechanisms underlying integrin activation remain poorly understood. The β subunit contains a PSI domain that is highly conserved across integrins and species, though its function is unknown. Integrin β subunits are cysteine-rich and endogenous thiol isomerase activity in integrin β3 has been reported. The PSI domain contains two CXXC sequences, the active site motif of protein disulfide isomerase (PDI). Based on this observation and the location of this domain at the knee region of the integrin, we hypothesized that integrin PSI domain has endogenous thiol isomerase function, which plays a key regulatory role in integrin conformation and function. Targeting the PSI domain may have therapeutic potential. Using reduced, denatured RNase, a recombinant murine integrin β3 PSI domain demonstrated endogenous PDI-like activity. This PDI-like activity was dose-dependently inhibited by the PDI inhibitor, bacitracin. Mutation of either CXXC motif within the integrin β3 PSI domain reduced PDI-like activity, while removal of both CXXC motifs completely abolished this activity. We developed unique mouse anti-mouse/anti-human β3 PSI domain monoclonal antibodies (anti-PSI mAbs) that inhibited the PDI-like activity of both the murine recombinant integrin β3 PSI domain and purified human platelet β3 integrin, in a dose-dependent manner. Interestingly, the anti-PSI mAbs blocked fibrinogen to human platelet β3 integrin in a cell free system. Furthermore, anti-PSI mAbs inhibited murine and human platelet aggregation in vitro and ex vivo and inhibited murine thrombus formation in vivo without significantly changing bleeding time or platelet count. In conclusion, we identified that the PSI domain has PDI function, is a fundamental regulator of platelet β3 integrin activation, and is a potential novel target for anti-thrombotic therapies. Since PSI domain is conserved in all integrin β subunits, our discovery may have broad implications for the role of integrins in cell biology of many human diseases.

Psi Domain of b3 Integrin Has Endogenous Thiol Isomerase Function and Is a Potential New Target for Anti-Thrombotic Therapy

AbstractAbstract 382 Background:Platelets are critical for maintaining hemostasis, but inappropriate platelet activation can lead to pathogenic thrombosis. Integrin aIIbb3, the most abundant protein on the platelet surface, is a key molecule for platelet aggregation and thrombus formation. The PSI domain of b3 integrin is highly conserved among different species but the function of the PSI domain in the integrin family has not been well defined and the role of this domain in hemostasis and thrombosis is poorly understood. It has been reported that b3 integrin possess PDI activity, which may play a role in integrin activation and platelet aggregation. However, whether the PSI domain of b3 integrin has PDI function is currently unknown. Methods:We generated recombinant protein of PSI domain of mouse b3 integrin. Mouse anti-mouse PSI domain mAbs were generated utilising b3 gene deficient mice (b3−/−) immunized with the recombinant protein. Antibody specificity was determined by flow cytometry and western blot. PDI activity assay of mouse PSI domain and native human b3 integrin was performed using reduced and denatured RNase (rdRNase). The effects of the mAbs on platelet function were measured in vitro using aggregometry and in vivo using intravital microscopy thrombosis model. Results:Analysis of the PSI domain of b3 integrin reveals that it contains two CXXC amino acid sequences (the active site motif of PDI), which are highly conserved in different species. Refolding of rdRNase assay showed that the PSI recombinant protein has endogenous PDI activity. Bacitracin, a well-known PDI inhibitor, inhibited PSI domain PDI function in a dose dependent manner. Four anti-PSI domain monoclonal antibodies (mAbs) were generated and showed different inhibitory effects on PDI function of the recombinant PSI domain and purified human platelet b3 integrin. In vitro and ex-vivo studies showed that anti-PSI antibodies inhibited mouse and human platelet aggregation. Using intravital microscopy we demonstrated that anti-PSI mAbs inhibited mouse platelet aggregation and thrombus formation in laser injury thrombosis model. Conclusions:To the best of our knowledge, this is the first time in which it has been demonstrated that the PSI domain of b3 integrin has endogenous PDI activity, which may play important roles in cell biology of platelets and other cells. Our data suggest that the PSI domain of b3 may be a new target in controlling platelet function and our mAbs may have potential in anti-thrombotic therapy. Disclosures:No relevant conflicts of interest to declare.

Initiation of Thrombus Formation: Novel Role for Thiol Isomerases

A fundamental component of protein biosynthesis is the intracellular generation or isomerization of disulfide bonds by thiol isomerases. Nature has apparently borrowed, or more accurately, hijacked this enzymatic machinery to regulate the extracellular initiation of thrombus formation in injured blood vessels. Protein disulfide isomerase, a prototypic thiol isomerase, is of critical import during in vivo arterial thrombus formation. This enzyme is secreted by the stimulated endothelium and activated platelets and is captured within the growing thrombus by b3 integrins, specifically aIIbb3 on platelets and avb3 on the endothelium. Inhibition of PDI blocks both platelet thrombus formation by preventing integrin activation and fibrin generation by preventing the expression of tissue factor activity. High throughput screening of chemical libraries has identified a series of flavonoids, including quercetin, isoquercetin and quercetin rutinoside, that are potent low molecular weight inhibitors of PDI in vitro. ERp5, a PDI-like thiol isomerase, is similarly secreted by platelets and plays a role in thrombus formation in vivo. Low molecular weight thiol isomerase inhibitors block platelet thrombus formation and fibrin generation in vivo and offer a novel target for anti-thrombotic therapy. The precise mechanism by which thiol isomerases control thrombus initiation remains to be unraveled.

GPR92/LPA5 lysophosphatidate receptor mediates megakaryocytic cell shape change induced by human atherosclerotic plaques

AimsOxidative processes and vascular inflammation underlying atherosclerosis lead to an accumulation of lysophosphatidic acid (LPA) molecules in the atheromatous intima. LPA, a platelet-activating component of human atherosclerotic plaques, possibly contributes to atherothrombus formation after plaque rupture. Human platelets express mRNA for the G protein-coupled receptors LPA1–7 that derive from megakaryocytes. The aim of our study was to identify the functional LPA receptor(s) in human platelets by silencing individual LPA receptors in megakaryocytic (MK) cells.Methods and resultsWe studied shape change of two human MK cell lines (Meg-01, Dami) by turbidometry, phase-contrast and scanning electron microscopy. They showed upon LPA stimulation a rapid, Rho-kinase-mediated shape change similar to that of human platelets. By qRT–PCR analysis we found expression of LPA1–7 in both cell lines; LPA4 and LPA5 were the most abundant receptor transcripts. In both Meg-01 and Dami cells, the rank order of activation by LPA species was similar to that found in platelets: alkyl-LPA 18:1 > alkyl-LPA 16:0 > acyl-LPA 18:1 >> alkyl-LPA 18:0. Knock-down of individual LPA receptors by siRNA showed that LPA-mediated activation of MK cells was mediated by LPA5, but not by LPA1–4,6,7. Importantly, we found that human atherosclerotic plaque and lipid-rich core induced shape change of Dami cells, and that this effect was inhibited after LPA5 silencing.ConclusionsOur findings indicate that LPA5 mediates LPA-induced shape change of MK cells and support its involvement in atherosclerotic plaque and lipid-rich core-mediated platelet activation. This receptor could be an attractive novel target for antithrombotic therapy.

Heat Shock Protein 47: A New Platelet Collagen Receptor

AbstractAbstract 158We recently reported that Heat shock protein 47 (Hsp47), a well established collagen binding protein, was present on the surface of human platelets. In the current study we demonstrate that anti-Hsp47 antibodies and a small molecule inhibitor of Hsp47 selectively prevent aggregation of platelets induced with collagen fibrils. Inhibition of Hsp47 was also found to reduce the size of thrombi produced upon perfusion of blood under arterial flow conditions over collagen in vitro. Inclusion of Integrilin, to prevent platelet-platelet interactions via fibrinogen, indicated that the reduced size of thrombi was due to fewer direct platelet-collagen interactions. Collagen-induced tyrosine phosphorylation of Syk, an early marker of signalling from the collagen receptor GPVI was unaffected by the Hsp47 inhibitor, suggesting that any contribution to platelet function by Hsp47 may be independent of GPVI-proximal signalling. Surface plasmon resonance analysis of recombinant his-tagged human Hsp47 revealed its ability to interact directly with collagen fibrils, an interaction that was disrupted by the small molecule inhibitor of Hsp47.Mouse platelets were also found to contain Hsp47, and aggregation induced with collagen was reduced by inhibitors of Hsp47. Infusion of Hsp47 inhibitor into mice increased tail bleeding times and volumes of blood lost, even in the absence of anticoagulant. This effect was likely due to inhibition of platelet function, since the thrombi formed in cremaster muscle arterioles using an intravital laser-injury model of thrombosis were smaller following administration of the Hsp47 inhibitor.Together these data indicate Hsp47 to function on the platelet surface as a collagen receptor that potentially represents a new target for antithrombotic therapy. Disclosures:No relevant conflicts of interest to declare.