Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains

Integrin αIIbβ3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbβ3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIb Calf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf- 1+ Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

Bruton’s Tyrosine Kinase Inhibitors Impair FcγRIIA-Driven Platelet Responses to Bacteria in Chronic Lymphocytic Leukemia

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton’s tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbβ3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbβ3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions.

TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Background Thrombus 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. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant 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. Results VWF 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. Conclusions This 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. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

Snake Venom Botrocetin Induces GPIb-Independent Platelet Aggregation

Abstract Introduction：Snake venom-derived botrocetin facilitates von Willebrand factor (VWF) binding to GPIbα, and has been used clinically for the detection of von Willebrand disease (VWD) and GPIb-related disorders. Botrocetin has also been widely used experimentally for the development and characterization of potential antithrombotic drugs targeting the GPIb-VWF axis. Although compelling evidence suggests GPIb is responsible for botrocetin-induced VWF binding and platelet aggregation, some reports suggest that botrocetin could induce platelet aggregation in some Bernard-Soulier syndrome (BSS) patients who lack a functional GPIb complex. However, the alternative mechanism for botrocetin-induced BSS platelet aggregation and the receptor(s) mediating this action are unclear. Methods: Botrocetin was purified from the lyophilized venom of Bothrops jararaca using ion-exchange column chromatography. Light transmission aggregometry assay was performed using platelet-rich plasma (PRP) from human, wild type (WT) mice, GPIbα-deficient mice, αIIbβ3-deficient mice and VWF-deficient mice, or CHO cells stably transfected with αIIbβ3 integrin. O-sialoglycoprotein endopeptidase (OSGE) was used to cleave the N-terminal extracellular domain of GPIbα. The binding of botrocetin, VWF and fibrinogen to platelets from WT or the gene-deficient mice were measured by flow cytometry. Antibodies against GPIbα (SZ2, NIT A) and integrin αIIbβ3 (abciximab, JON/A, M1, PSI E1) were used to investigate the binding site of botrocetin. Perfusion chamber assay was used to measure thrombus formation under different shear stresses. Results: We discovered that botrocetin induced aggregation of human platelets lacking the N-terminal extracellular domain of GPIbα and platelets from GPIbα-deficient mice in the presence of VWF. This VWF-dependent, GPIbα-independent platelet aggregation induced by botrocetin was inhibited by αIIbβ3 antagonists. Botrocetin also induced aggregation of CHO cells stably transfected with αIIbβ3 in VWF-dependent manner. Further experiments with gel-filtered platelets showed that botrocetin competitively bound to the ligand-binding area exposed on αIIbβ3 and blocked fibrinogen and other ligands from binding to the active state of αIIbβ3 in the absence of VWF. Botrocetin inhibited platelet aggregation and thrombus formation in VWF-deficient mice. Conclusion: Integrin αIIbβ3 is the alternative receptor that mediates VWF-dependent, GPIb-independent platelet aggregation induced by botrocetin. However, via targeting αIIbβ3, botrocetin itself inhibits platelet aggregation in the absence of VWF. These results demonstrate versatility in the mechanism of botrocetin, which may provide snakes containing this toxin the adaptability necessary to aggregate platelets/thrombocytes of different prey or predators. Our data reveals a previously unknown role of botrocetin in the integrin-VWF interaction and also provides insight into developing new antithrombotic drugs that target the active conformation of integrin αIIbβ3. The target switching of botrocetin between GPIb-VWF and αIIbβ3-VWF may explain the possible misdiagnosis of the GPIb-related congenital disorders evaluated by botrocetin. Disclosures No relevant conflicts of interest to declare.

Aβ1–40-Induced Platelet Adhesion Is Ameliorated by Rosmarinic Acid through Inhibition of NADPH Oxidase/PKC-δ/Integrin αIIbβ3 Signaling

In platelets, oxidative stress reportedly increases platelet adhesion to vessels, thus promoting the vascular pathology of various neurodegenerative diseases, including Alzheimer’s disease. Recently, it has been shown that β-amyloid (Aβ) can increase oxidative stress in platelets; however, the underlying mechanism remains elusive. In the present study, we aimed to elucidate the signaling pathway of platelet adhesion induced by Aβ1–40, the major form of circulating Aβ, through Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting analysis. Additionally, we examined whether rosmarinic acid (RA), a natural polyphenol antioxidant, can modulate these processes. Our results show that Aβ1–40-induced platelet adhesion is mediated through NADPH oxidase/ROS/PKC-δ/integrin αIIbβ3 signaling, and these signaling pathways are significantly inhibited by RA. Collectively, these results suggest that RA may have beneficial effects on platelet-associated vascular pathology in Alzheimer’s disease.

Berberine and Its Main Metabolite Berberrubine Inhibit Platelet Activation Through Suppressing the Class I PI3Kβ/Rasa3/Rap1 Pathway

Background: Berberine (BBR), a natural product, was reported to inhibit platelet aggregation; however, the molecular mechanisms remain unclear. This study aims to investigate the effects and mechanisms of BBR in inhibiting platelet activation and thrombus formation.Methods: Flow cytometry, immunofluorescence, and Western blot were used to determine the inhibitory effects and mechanisms of BBR and its main metabolite berberrubine (M2) on platelet activation in vitro and ex vivo. Purified integrin αIIbβ3, class I PI3K kit, and molecular docking were used to identify the possible targets of BBR and M2. A carrageenan-induced mouse thrombosis model was used to evaluate the effects of BBR on thrombus formation in vivo.Results:In vitro, BBR and M2 significantly inhibited ADP-induced integrin αIIbβ3 activation, reduced the level of P-selectin on the platelet membrane, and suppressed the binding of fibrinogen to the platelets. In this process, BBR and M2 greatly suppressed the PI3K/Akt pathway and inhibited Rasa3 membrane translocation and Rap1 activation. Furthermore, BBR and M2 selectively inhibited class I PI3Kβ, perhaps through binding to its active site. The activities of BBR were stronger than those of M2. After oral administration, BBR significantly inhibited the PI3K/Akt pathway and Rap1 activation and suppressed ADP-induced platelet activation and carrageenan-induced thrombosis in mice without prolonging bleeding time.Conclusions: We reveal for the first time the possible targets and mechanisms of BBR and M2 in inhibiting platelet activation. Our research may support the future clinical application of BBR as an antiplatelet drug in the prevention or treatment of thrombotic diseases.

The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis

Hyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Here, a human medical examination report showed that elevated human plasma Hcy levels were positively correlated with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation both in acute and chronic HHcy ApoE-/- mice. Upon Hcy administration in C57BL/6J mice, platelet aggregation, spreading, and clot retraction were markedly promoted. More importantly, homocysteine (Hcy) increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine (PS) exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE-/- mice. This study identifies a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently to exaggerate thrombosis. This study reveals an innovative approach to treat HHcy-related thrombotic diseases.