αIIbβ3 Binding Research Articles

#1593 ARGDRR, a novel disintegrin, attenuates AKI to CKD-related renal fibrosis

Abstract Background and Aims Acute kidney injury (AKI) can lead to progress renal fibrosis, resulting in chronic kidney disease (CKD) and end stage renal disease (ESRD). Nevertheless, the underlying pathological mechanisms remain unclear. Platelet activation has been demonstrated as one of the important factors involved in the pathophysiology of AKI, and studies show the therapeutic potential for using platelet inhibitors or antagonists in ischemia-reperfusion (IR)-induced AKI models. However, drug-induced side effects, including thrombotic microangiopathies (TMA)-related kidney damage, thrombocytopenia and bleeding, hinder clinical application in AKI or CKD treatment. Our newly developed dual effect disintegrin, ARGDRR, is derived from snake venom exert a high affinity to αIIbβ3 and αvβ3 binding affinity without causing bleeding and other thrombosis-related side effects. Method We performed a unilateral ischemia-reperfusion injury (UIRI) model to investigate integrin αIIb(Itga2b) and β3(Itgb3) mRNA levels in the injured kidney for 15 days. For exploring the therapeutic efficiency of ARGDRR in AKI, we administered ARGDRR post-ischemic injury and measured platelet activation after 24 hours using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) for Itgb3, Lcn2, and CD42b. For exploring the therapeutic efficiency of ARGDRR in CKD progression, we administrated ARGDRR daily for 15 days after IRI with nephrectomy, and examined the difference of renal dysfunction, renal fibrosis, and cell death compared to sham group. Results We found that platelet activation with progressively increased mRNA level of platelet glycoprotein IIb-GPIIIa (Itga2b and Itgb3) in the UIRI kidney over time. After 24 hr of ischemic AKI, ARGDRR administration reduced Lcn2 and Itgb3 mRNA level and CD42b positive area in the kidney, indicating its efficacy in protecting the kidneys by inhibiting platelet activation and aggregation. In AKI to CKD transition, serum creatine and blood urea nitrogen (BUN) were significantly lower both in efficacious and high dose of ARGDRR administration group. ARGDRR significantly reduced CD42b expression and increased LTL positive area. Also, ARGDRR administration alleviated renal fibrosis, cell apoptosis and senescence-related molecules p21 and Ctgf expression in the AKI to CKD transition. Conclusion Our results have successfully proved our hypothesis that disintegrin-ARGDRR has the potential for renal protection in the transition of AKI to CKD through inhibition of platelet activation and aggregation during acute injury.

CD40L Activates Platelet Integrin αIIbβ3 by Binding to the Allosteric Site (Site 2) in a KGD-Independent Manner and HIGM1 Mutations Are Clustered in the Integrin-Binding Sites of CD40L.

CD40L is expressed in activated T cells, and it plays a major role in immune response and is a major therapeutic target for inflammation. High IgM syndrome type 1 (HIGM1) is a congenital functional defect in CD40L/CD40 signaling due to defective CD40L. CD40L is also stored in platelet granules and transported to the surface upon platelet activation. Platelet integrin αIIbβ3 is known to bind to fibrinogen and activation of αIIbβ3 is a key event that triggers platelet aggregation. Also, the KGD motif is critical for αIIbβ3 binding and the interaction stabilizes thrombus. Previous studies showed that CD40L binds to and activates integrins αvβ3 and α5β1 and that HIGM1 mutations are clustered in the integrin-binding sites. However, the specifics of CD40L binding to αIIbβ3 were unclear. Here, we show that CD40L binds to αIIbβ3 in a KGD-independent manner using CD40L that lacks the KGD motif. Two HIGM1 mutants, S128E/E129G and L155P, reduced the binding of CD40L to the classical ligand-binding site (site 1) of αIIbβ3, indicating that αIIbβ3 binds to the outer surface of CD40L trimer. Also, CD40L bound to the allosteric site (site 2) of αIIbβ3 and allosterically activated αIIbβ3 without inside-out signaling. Two HIMG1 mutants, K143T and G144E, on the surface of trimeric CD40L suppressed CD40L-induced αIIbβ3 activation. These findings suggest that CD40L binds to αIIbβ3 in a manner different from that of αvβ3 and α5β1 and induces αIIbβ3 activation. HIGM1 mutations are clustered in αIIbβ3 binding sites in CD40L and are predicted to suppress thrombus formation and immune responses through αIIbβ3.

Altered fibrinogen γ-chain cross-linking in mutant fibrinogen-γΔ5 mice drives acute liver injury

BackgroundHepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury. ObjectivesWe sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbβ3 binding domain in APAP-induced liver injury. MethodsMice expressing mutant fibrinogen incapable of engaging integrin αIIbβ3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally). ResultsWe observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbβ3. ConclusionThe results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.

TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

BackgroundThrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors.AimWe aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation.MethodsRecombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma.ResultsVWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1.Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice.ConclusionsThis study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. DisclosuresRuf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

Mechanisms Linking Fibrin(ogen) Structure/Function to Tumor Metastasis

Previous studies established that the platelet/fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (NK) cells. However, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. It also remains to be determined if the link between fibrin(ogen) and innate tumor immunity is relevant to tumor growth in some contexts, or is strictly limited to early events important in metastasis.To test the hypothesis that fibrin(ogen) promotes metastasis by distinct mechanisms linked to specific structure/function interactions with several cell types, we performed experimental metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. Expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)-inflammatory cell interactions mediated by αMβ2 in metastasis. To directly determine the importance of thrombin-mediated fibrin polymerization in metastasis, we analyzed metastatic potential in FibAEK mice, which carry a form of fibrinogen essentially “locked” in the soluble state due to a germ-line mutation in the Aα chain thrombin cleavage site. Metastatic potential in FibAEK mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymer formation in this process. However, the FibAEK mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains some significant prometastatic properties. In order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and FibγΔ5 mice, carrying a form of fibrinogen lacking the γ chain binding motif for the platelet integrin αIIbβ3. Surprisingly, this mutation had no impact on metastatic potential. Given the established importance of platelets in metastasis, these findings suggest that fibrin is capable of platelet stabilization through mechanism(s) independent of this αIIbβ3 binding motif.Fibrin(ogen) appears to be important in metastasis across multiple tumor types, but its role in tumor growth is context dependent. Previous studies established that fibrin(ogen) promotes the growth of colonic adenocarcinoma in mice. To determine if fibrin(ogen) is mechanistically coupled to NK cell functions in this context, we depleted NK cells in cohorts of Fib-/- and control mice with an anti-NK1.1 antibody prior to subcutaneous inoculation of MC38 cells (a C57Bl/6-derived colonic adenocarcinoma). Depletion of NK cells had no impact on tumor growth in Fib+ mice. In mice with intact NK cells (i.e., inoculated with a nonspecific IgG), fibrinogen deficiency significantly limited tumor growth. However, depletion of NK cells resulted in a restoration of tumor growth in Fib-/- mice that was comparable to that observed in Fib+ mice.Together, these studies suggest that both fibrin and fibrinogen play a significant role in metastasis. In addition to interactions with platelets, fibrin(ogen) appears to support metastasis through leukocyte interactions mediated by αMβ2. These studies also reveal an important context where fibrin(ogen) promotes tumor growth via mechanisms coupled to NK cell functions, opening the door to the possibility that targeting these interactions could provide a means to limit the progression of established tumors. DisclosuresNo relevant conflicts of interest to declare.

Utilizing CRISPR-CAS9 Gene Editing Technology in Human Pluripotent Stem Cells to Study Platelet Integrin αIIbβ3 Function

Integrins are a family of cell surface receptors that transmit signals bidirectionally across plasma membranes. Cellular agonists induce 'inside-out' signals that cause ligand binding to integrins; in turn, this causes integrin-mediated 'outside-in' signals that control cellular functions such as adhesion and migration. The platelet integrin αIIbβ3 has served as a paradigm for deciphering these bidirectional signaling mechanisms. Moreover, site-directed mutagenesis has been essential for dissecting integrin structure-function relationships. For platelet integrins such as αIIbβ3, the structure-function consequences of the site-directed mutations have previously been studied in epithelial cell lines, such as Chinese hamster ovary (CHO) cells. Only recently has technology advanced sufficiently to enable study of platelet integrins in their normal cellular environment, the platelet-producing megakaryocyte. The advent of human induced pluripotent stem cells (iPSC), coupled with the ability to gene edit these cells using the CRISPR-CAS9 technology provides a way to study αIIbβ3 mutants in megakaryocytes. Following platelet stimulation, αIIbβ3 rapidly undergoes a global rearrangement in which a clasp composed of its extracellular stalk, transmembrane, and membrane-proximal cytoplasmic domains is disrupted causing the αIIbβ3 headpiece to open exposing a ligand binding site. Using computational methods, we predicted mutations that would destabilize the stalk domain, thereby causing αIIbβ3 activation. These predictions were confirmed by showing that the mutations were indeed able to cause constitutive αIIbβ3 activation in CHO cells (Donald et al, J. Biol. Chem. 2010; Tan et al, Biochemistry 2019). To translate these findings to human cells and compare the functional consequences of the mutations when endogenously expressed in iPSC-derived megakaryocytes, we chose a highly activating V760A missense mutation located in the stalk region of the αIIb subunit. Using an established control iPSC line designated CHOPWT14, both heterozygous and homozygous V760A missense mutations were created in the ITGA2B gene encoding αIIb using our CRISPR-CAS9 protocol (Maguire et al. Current Protocols in Stem Cell 2019). The generation of these mutant cell lines from a genetically identical control cell line eliminates the heterogeneity and variability in downstream applications that was observed when using lines of different genetic backgrounds. To ensure genetic identity of the parent and mutant lines, copy number variation or CNV analyses were performed and shown to be identical in all lines. To study ligand binding of αIIbβ3 expressing the V760A mutation in iPSC-derived megakaryocytes, the isogenic control and mutant lines were differentiated to blood using our serum- and stromal-free directed differentiation protocol (Mills et al. Methods in Mol. Biol. 2014). Briefly, the differentiation of human iPSCs to blood follows primitive hematopoiesis which recapitulates the developmental stages observed in the embryo of primitive streak formation, mesoderm specification, and hematopoietic development, including megakaryocytes. To measure conformational change in αIIbβ3, the activation-dependent monoclonal antibody PAC-1 was used as a surrogate for fibrinogen. By analyzing CD41+CD42b+ megakaryocytes by flow cytometry, PAC-1 was shown to bind constitutively to 23.4% and 26.0% of the megakaryocytes expressing heterozygous and homozygous V760A mutations, respectively, compared to 9.04% of control megakaryocytes. The specificity of PAC-1 binding was confirmed by a reduction in binding to <1% following pretreatment with EDTA. In addition, thrombin stimulation increased PAC-1 binding to >65% in all lines, indicating normal overall αIIbβ3 function. Essentially identical results were obtained when FITC-fibrinogen was used instead of PAC-1. These data show that 1) structure-function studies of computationally identified mutations confirmed in CHO cells can be analyzed using human iPSC-derived megakaryocytes, 2) mutations shown to be highly active in CHO cells appear to be constrained or less constitutively active in human megakaryocytes, and 3) more in-depth analyses of platelet integrin structure-function relationships will be possible using human megakaryocytes. Disclosures No relevant conflicts of interest to declare.

Platelet hyperactivation in multiple myeloma is also evident in patients with premalignant monoclonal gammopathy of undetermined significance

Thrombotic events are common in patients with multiple myeloma (MM), smouldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS). Previous studies have indicated platelet hyperactivation as a feature of thrombotic risk in MM, but there is a dearth of data in MGUS. In the present study, multiparameter analysis of platelet activation and responsiveness was investigated by flow cytometry in patients with MGUS, SM/MM and healthy controls (HCs). The median platelet surface CD63 levels, annexin V and PAC-1 antibody (specific for activated integrin αIIbβ3) binding were significantly elevated in patients with MGUS versus the HCs. These markers were also elevated in SM/MM, but not significantly. In all, 74% of MGUS and 38% of SM/MM patients had one or more elevated marker of platelet activation, compared to 19% of the HCs. Marker-specific hyporesponsiveness of platelets to agonist [adenosine diphosphate (ADP), thrombin receptor-activating peptide 6] stimulation in vitro was observed, with significantly reduced surface levels of P-selectin in response to ADP in patients with MGUS. Platelet-leucocyte aggregates were not altered in patients, while platelet-associated immunoglobulins were elevated in a subset of patients. Overall, we found that platelet hyperactivation is prevalent in both MGUS and SM/MM patients and is potentially related to hyporesponsiveness. These observations suggest that further investigation of the predictive and prognostic value of platelet hyperactivation in such patients is warranted.

Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2.

Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Ibα-dependent platelet 'priming' induces integrin αIIbβ3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet αIIbβ3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated αIIbβ3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil crosstalk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.

A Novel αIIbβ3 Antagonist from Snake Venom Prevents Thrombosis without Causing Bleeding.

Life-threatening thrombocytopenia and bleeding, common side effects of clinically available αIIbβ3 antagonists, are associated with the induction of ligand-induced integrin conformational changes and exposure of ligand-induced binding sites (LIBSs). To address this issue, we examined intrinsic mechanisms and structure–activity relationships of purified disintegrins, from Protobothrops flavoviridis venom (i.e., Trimeresurus flavoviridis), TFV-1 and TFV-3 with distinctly different pro-hemorrhagic tendencies. TFV-1 with a different αIIbβ3 binding epitope from that of TFV-3 and chimeric 7E3 Fab, i.e., Abciximab, decelerates αIIbβ3 ligation without causing a conformational change in αIIbβ3, as determined with the LIBS antibody, AP5, and the mimetic, drug-dependent antibody (DDAb), AP2, an inhibitory monoclonal antibody raised against αIIbβ3. Consistent with their different binding epitopes, a combination of TFV-1 and AP2 did not induce FcγRIIa-mediated activation of the ITAM–Syk–PLCγ2 pathway and platelet aggregation, in contrast to the clinical antithrombotics, abciximab, eptifibatide, and disintegrin TFV-3. Furthermore, TFV-1 selectively inhibits Gα13-mediated platelet aggregation without affecting talin-driven clot firmness, which is responsible for physiological hemostatic processes. At equally efficacious antithrombotic dosages, TFV-1 caused neither severe thrombocytopenia nor bleeding in FcγRIIa-transgenic mice. Likewise, it did not induce hypocoagulation in human whole blood in the rotational thromboelastometry (ROTEM) assay used in perioperative situations. In contrast, TFV-3 and eptifibatide exhibited all of these hemostatic effects. Thus, the αIIbβ3 antagonist, TFV-1, efficaciously prevents arterial thrombosis without adversely affecting hemostasis.

Genetic Absence of Integrin alpha2b Improves Survival of Tissue Factor Pathway Inhibitor Null Mice but Results in Hydrocephalus Formation

Introduction: Genetic deletion of Tissue Factor Pathway Inhibitor (TFPI) exon 4, encoding its Kunitz 1 (K1) domain, results in complete intrauterine lethality (PMID 9242522). TFPI K1 null mice (Tfpi-/-) are born live if Tissue Factor expression is reduced or in the complete absence of Protease Activated Receptor-4 (Par4), and these exhibit a normal life span without overt signs of thrombosis (PMID 15598816, 25954015). Based on these data, it has been postulated that modulation of thrombin-dependent platelet activation by TFPI is essential for survival. Platelet activation results in a number of downstream events. Of these, platelet aggregation via the integrin receptor αIIbβ3 is considered to have a key role in hemostasis and could participate in thrombotic pathology in the absence of TFPI. Binding of αIIbβ3 to its ligands also mediates critical interactions of platelets with endothelial cells, leukocytes and other cell types. In this work, we have investigated whether modulation of platelet activity via genetic absence of integrin receptor αIIbβ3 confers protection and allows generation of adult Tfpi-/- mice. Methods: Tfpi+/- αIIb-/- mice were generated by breeding Tfpi+/- and integrin αIIb-/- mice and identified by PCR-based genotyping of tissues obtained by tail biopsies. Tfpi+/- αIIb-/- intercrosses served as the experimental cross. Pups were genotyped at the time of wean, around 4 weeks of age. In some experiments, surgeries were performed to analyze pregnancies at 18.5 days post coitum (dpc). Embryos and placentae were observed under the dissecting microscope and any phenotypic abnormalities were noted. Presence of heart beats and limb movements were used to identify live embryos. Embryos and placentae were fixed in zinc-formalin and embedded in paraffin for sectioning and histological analysis. T1 weighted Magnetic Resonance Images were acquired on a 9.4T scanner to measure cerebral ventricle sizes of Tfpi-/- αIIb-/- and littermate control mice. Ventricular regions of interest (ROI) were drawn on each image slice from which total ventricular volume was computed. These mice were later perfused with 4% paraformaldehyde for collection of organs and histological analysis. Results: We analyzed 122 pups from intercrosses of Tfpi+/- αIIb-/- mice and observed a genetic distribution 39 Tfpi+/+, 77 Tfpi+/- and 6 Tfpi-/- (25% or 31 Tfpi-/- were expected, 5% observed, 95% CI 1.8 to 10.4%). Thus, genetic absence of αIIb results in incomplete rescue of Tfpi-/- mice (P< 0.000002, Χ2 GOF) with only ~20% surviving past embryonic development to 4 weeks of age. These data contrast with 40% Tfpi-/- offspring surviving in the absence of Par4 (Tfpi+/- Par4-/- intercrosses: 23 Tfpi+/+, 39 Tfpi+/- and 8 Tfpi-/-; PMID 25954015). Thus, the absence of αIIb is much less effective than the absence of Par4 in allowing early survival of TFPI null mice (P < 1.9E-09; Χ2 test of independence). We compared survival of Tfpi-/- αIIb-/- offspring close to term of pregnancy (18.5 dpc) and at 4 weeks of age. TFPI null embryos were found at reduced frequency at 18.5 dpc (12 Tfpi+/+, 21 Tfpi+/- and 5 Tfpi-/-; 25% Tfpi-/- expected, 13% observed, 95% CI 4.4 to 28.1%), but even fewer survived the trauma of birth (P<0.0004, Χ2 test of independence). Of the 6 Tfpi-/- αIIb-/- pups found at 4 weeks of age, 3 died by 9 weeks of age. Dome shaped heads indicative of hydrocephalus or histological evidence of hydrocephalus was noted in the pups that died. Surviving mice were observed for 7 months to 1 year of age and imaged with MRI. Comparison of ventricular volumes between Tfpi-/- mice and Tfpi+/- controls demonstrated a higher volume in Tfpi-/- mice (39.3 ± 18.3 versus 5.3 ± 2.2 mm3; P=0.08). Both Tfpi-/- and Tfpi+/- mice were αIIb-/- in this experiment. Thus, αIIb in not involved in hydrocephalus formation. Hydrocephalus formation in Tfpi-/- mice was confirmed through serial histological sections of the brain (Figure 1). Conclusions: Our data demonstrates that genetic absence of αIIb improves survival of TFPI null mice, but to a much lesser extent than the genetic absence of Par4. Thus, the critical role of Par4 in the demise of TFPI null mice is unlikely to be primarily through excessive platelet aggregation. We further show that TFPI null pups exhibit varying degrees of hydrocephalus formation. While the mechanism of hydrocephalus formation in the absence of TFPI remains unclear, our results demonstrate a critical role of TFPI in the brain. Disclosures No relevant conflicts of interest to declare.

A Multiscale Model for Recruitment Aggregation of Platelets by Correlating with In Vitro Results.

We developed a multiscale model to simulate the dynamics of platelet aggregation by recruitment of unactivated platelets flowing in viscous shear flows by an activated platelet deposited onto a blood vessel wall. This model uses coarse grained molecular dynamics (CGMD) for platelets at the microscale and dissipative particle dynamics (DPD) for the shear flow at the macroscale. Under conditions of relatively low shear, aggregation is mediated by fibrinogen via αIIbβ3 receptors. The binding of αIIbβ3 and fibrinogen is modeled by a molecular-level hybrid force field consisting of Morse potential and Hooke law for the nonbonded and bonded interactions, respectively. The force field, parametrized in two different interaction scales, is calculated by correlating with the platelet contact area measured in vitro and the detaching force between αIIbβ3 and fibrinogen. Using our model, we derived, the relationship between recruitment force and distance between the centers of mass of two platelets, by integrating the molecular-scale inter-platelet interactions during recruitment aggregation in shear flows. Our model indicates that assuming a rigid-platelet model, underestimates the contact area by 89% and the detaching force by 93% as compared to a model that takes into account the platelet deformability leading to a prediction of a significantly lower attachment during recruitment. The molecular-level predictive capability of our model sheds a light on differences observed between transient and permanent platelet aggregation patterns. The model and simulation framework can be further adapted to simulate initial thrombus formation involving multiple flowing platelets as well as deposition and adhesion onto blood vessels.

Screening of pure synthetic coating substrates for induced pluripotent stem cells and iPSC-derived neuroepithelial progenitors with short peptide based integrin array

Simple and pure synthetic coating substrates are needed to overcome the disadvantages of traditional coating products like animal derived Matrigel in stem cell research. Since integrins are of great importance in cell adhesion and cell-ECM communication, in this study, a commercially available integrin array established by synthetic integrin binding peptides is used to screen coating substrates for iPSCs and NEPs. The results showed that binding peptides of integrin α5β1, αVβ1, αMβ2 and αIIbβ3 supported cell adhesion of iPSCs, while α5β1, αVβ1 and αIIbβ3 binding peptides supported NEPs adhesion. Additionally, integrin α5β1 binding peptide was revealed to support rapid expansion of iPSCs and iPSC-derived NEPs, as well as the process of NEPs generation, with equal efficiency as Matrigel. In this work, we demonstrated that by supporting stem cell growth in an integrin dependent manner, the integrin array and coating system has the potential to develop more precise and efficient systems in neurological disease modeling.

A Heterozygous Mutation of G-Protein-Coupled Receptor 25 in a Family with Inherited Thrombocytopenia and Thrombosis

BackgroundInherited thrombocytopenia is associated with bleeding tendency and no association with thrombotic tendency has been reported. We found a Japanese family with autosomal dominant form of thrombocytopenia associated with arterial and venous thrombosis.AimsTo identify a causative gene for hereditary thrombocytopenia with thrombosis.MethodsA propositus was a 42-year-old Japanese woman with thrombocytopenia and deep vein thrombosis (DVT). She had normal activities of antithrombin, protein C, and protein S, and negative results for lupus anticoagulant and anti-cardiolipin antibodies. She has suffered from thrombocytopenia (20 - 50 x 109/L) for at least 20 years. Six members among her family pedigree had thrombocytopenia, and also suffered from thrombosis including mesenteric artery thrombosis, young-onset myocardial infarction, femoral artery thrombosis, repeated cerebral infarction, DVT, and pulmonary embolism. Importantly, the family members with these thrombotic disorders had exclusively thrombocytopenia. Whole exome sequencing was performed with venous blood from the 6 affected and 6 non-affected family members to identify a causative gene for thrombocytopenia.ResultsWe identified a heterozygous change (c.764G>T:p.G255V) in GPR25 gene, an uncharacterized G protein-coupled receptor. The mutation was completely linked with thrombocytopenia. The mutated residue was located at the transmembrane domain of GPR25. We generated anti-GPR25 monoclonal antibodies and confirmed that GPR25 was expressed on the platelet surface. The patient's platelets showed the same level of PAC1 (a monoclonal antibody recognizing the active conformation of integrin αIIbβ3) binding and P-selectin expression as platelets from healthy subjects. However, ADP-induced aggregation of patient's washed platelets was enhanced as compared with normal washed platelets. Moreover, the patient's platelets had an increase in thrombin + collagen-induced and A23187-induced Annexin V binding compared with platelets from healthy subjects.ConclusionWe identified a novel gene mutation responsible for inherited thrombocytopenia and thrombosis that may induce platelet hyper-reactivity. DisclosuresNo relevant conflicts of interest to declare.

Norcantharidin, a clinical used chemotherapeutic agent, acts as a powerful inhibitor by interfering with fibrinogen-integrin αIIb β3 binding in human platelets.

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin αII bβ3, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin αII bβ3 activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β3, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin αII bβ3‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.

Soluble fibrin causes an acquired platelet glycoprotein VI signaling defect: implications for coagulopathy

Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through Gαq was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the αIIb β3 antagonist GR144053, indicating negligible roles for GPVI shedding or αIIb β3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in >50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90nm soluble fibrin, far exceeding ~1nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

An αIIbβ3 antagonist prevents thrombosis without causing Fc receptor γ‐chain IIa‐mediated thrombocytopenia

Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin αIIb β3 . Objective To explore the correlation between αIIb β3 binding epitopes and induction of DDAb binding to conformation-altered αIIb β3 , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of αIIb β3 conformational change and platelet aggregation in the presence of AP2, an IgG1 inhibitory mAb raised against αIIb β3 . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the αIIb β-propeller domain of αIIb β3 , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of αIIb β3 as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested αIIb β3 antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

Strong Binding of Platelet Integrin \u03b1IIb\u03b23 to Fibrin Clots: Potential Target to Destabilize Thrombi

The formation of platelet thrombi is determined by the integrin αIIbβ3-mediated interactions of platelets with fibrinogen and fibrin. Blood clotting in vivo is catalyzed by thrombin, which simultaneously induces fibrinogen binding to αIIbβ3 and converts fibrinogen to fibrin. Thus, after a short time, thrombus formation is governed by αIIbβ3 binding to fibrin fibers. Surprisingly, there is little understanding of αIIbβ3 interaction with fibrin polymers. Here we used an optical trap-based system to measure the binding of single αIIbβ3 molecules to polymeric fibrin and compare it to αIIbβ3 binding to monomeric fibrin and fibrinogen. Like αIIbβ3 binding to fibrinogen and monomeric fibrin, we found that αIIbβ3 binding to polymeric fibrin can be segregated into two binding regimes, one with weaker rupture forces of 30–60 pN and a second with stronger rupture forces >60 pN that peaked at 70–80 pN. However, we found that the mechanical stability of the bimolecular αIIbβ3-ligand complexes had the following order: fibrin polymer > fibrin monomer > fibrinogen. These quantitative differences reflect the distinct specificity and underlying molecular mechanisms of αIIbβ3-mediated reactions, implying that targeting platelet interactions with fibrin could increase the therapeutic indices of antithrombotic agents by focusing on the destabilization of thrombi rather than the prevention of platelet aggregation.

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3.

Binding of soluble fibrinogen to the activated conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3 and make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbβ3 conformers at the interface between the αIIb β-propeller domain and the β3 βI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbβ3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide-αIIbβ3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbβ3 that dissociate in the 60-120 pN range. The Gibbs free energy profiles of the αIIbβ3-peptide complexes revealed that the overall stability of the αIIbβ3-cRGDFK complex was comparable with that of the αIIbβ3-γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbβ3-fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbβ3 with immobilized fibrinogen and fibrin.

αIIbβ3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible

AbstractPlatelet integrin receptor αIIbβ3 supports platelet aggregation by binding fibrinogen. The interaction between the fibrinogen C-terminal γ-chain peptide composed of residues γ-404-411 (GAKQAGDV) and the Arg-Gly-Asp (RGD) binding pocket on αIIbβ3 is required for fibrinogen-mediated platelet aggregation, but data suggest that other ancillary binding sites on both fibrinogen and αIIbβ3 may lead to higher-affinity fibrinogen binding and clot retraction. To identify additional sites, we analyzed the ability of platelets and cells expressing normal and mutant αIIbβ3 to adhere to an immobilized fibrinogen plasmin fragment that lacks intact γ-404-411 ('D98'). We found the following: (1) Activated, but not unactivated, platelets adhere well to immobilized 'D98.' (2) Cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3 or αVβ3, adhere well to 'D98.' (3) Monoclonal antibodies 10E5 and 7E3 inhibit the adhesion to 'D98' of activated platelets and cells expressing constitutively active αIIbβ3, as do small-molecule inhibitors that bind to the RGD pocket. (4) EDTA paradoxically induces normal αIIbβ3 to interact with 'D98.' Because molecular modeling and molecular dynamics simulations suggested that the αIIb L151-D159 helix may contribute to the interaction with 'D98,' we studied an αIIbβ3 mutant in which the αIIb 148-166 loop was swapped with the corresponding αV loop; it failed to bind to fibrinogen or 'D98.' Our data support a model in which conformational changes in αIIbβ3 and/or fibrinogen after platelet activation and the interaction between γ-404-411 and the RGD binding pocket make new ancillary sites available that support higher-affinity fibrinogen binding and clot retraction.

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level.