Thiol Isomerase Activity of β 3 Integrin Psi Domain and L33P Polymorphism: Implications in Blood Coagulation and Anti-Thrombotic Therapy

Background: Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the "knee" region. Our lab recently discovered that two "CXXC" motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood, 2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo 10-30 fold more potent than their effects in vitro under anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear. Methods/Results: To investigate the role of PSI domain in blood coagulation, we first employed thromboelastography (TEG) to compare our anti-PSI domain mAbs with other anti-β3 mAbs that do not directly bind to β3 PSI domain. Results show that anti-PSI domain mAbs inhibited blood coagulation in human and murine whole blood or platelet-rich plasma (PRP) significantly more than anti-αIIbβ3 antibodies JAN-D1, M1, and Abciximab precursor 7E3. To address whether the L33P polymorphism affects PSI domains thiol isomerase activity, we generated L33P PSI domain via site-directed mutagenesis in E. coli. Using a scrambled RNase assay, we found that L33P polymorphism enhanced thiol-isomerase activity relative to WT PSI domain. We further corroborated these findings through an insulin β chain reduction assay, and a MPB (N-Maleimidopropionyl-biocytin) western blot assay, which quantifies thiol isomerase activity through MPB binding to free thiols that have not been oxidized into RNase. Interestingly, TEG results show that recombinant human PSI domain enhanced blood coagulation in platelet-microparticle (PMP) free plasma, which was generated through high-speed centrifugation (17,000 x g) of platelet poor plasma (PPP)for 15 minutes that removed residual platelets and microparticles . Conclusion: We have discovered a novel role of integrin β3 PSI domain in blood coagulation, which is enhanced by the L33P polymorphism (HPA-1b). These data highlight the β3 PSI domain as a suitable therapeutic target for its roles in both platelet adhesion/aggregation, and blood coagulation. Furthermore, these data may explain the increased risk of CVD such as myocardial infarction and deep vein thrombosis for individuals with the L33P polymorphism. Disclosures No relevant conflicts of interest to declare.

Application of a Simple Microfluidic Chip Analysis Technology to Evaluate the Inhibitory Role of Protocatechuic Acid on Shear-Induced Platelet Aggregation

This study aimed to develop a simple microfluidic chip analysis technology to study the inhibitory effect of protocatechuic acid on shear-induced platelet aggregation. The microfluidic chip designed in this study simulates 80% fixed narrow microchannels. This microchannel narrow model uses the finite element analysis module of the three-dimensional modeling software solidwork to analyze fluid dynamic behavior. Blood treated with protocatechuic acid at 1, 2, 4, 8, or 16 µg/mL was passed through the microchannel stenosis model at a shear rate of 10,000 s−1. The platelet adhesion and aggregation behaviors were then measured using fluorescence microscopy and observed in real time. Simultaneously, the antiplatelet aggregation effect of protocatechuic acid was analyzed using thromboelastography and photoelectric turbidimetry. The designed stenosis model of the microfluidic chip can produce a gradient of fluid shear rate, and the gradient of fluid shear rate can induce platelet aggregation. Under this model, the degree of platelet adhesion and aggregation increased as the shear rate increased. In the experimental concentration range of 0–8 µmol/mL, protocatechuic acid exerted a concentration-dependent inhibition of platelet aggregation. In contrast, thromboelastography and photoelectric turbidimetry failed to demonstrate an inhibitory effect. The microfluidic chip analysis technology developed in this study can be used to study the effect of protocatechin in inhibiting platelet aggregation induced by shear rate in vitro. This technology is simple to operate and can be used as a new type of antiplatelet aggregation analysis technology for screening studies of novel potential antiplatelet aggregation drugs.

Microfluidic chip grafted with integrin tension sensors for evaluating the effects of flowing shear stress and ROCK inhibitor on platelets

Integrins are key players in platelet adhesion and aggregation. Integrin molecular tensions, the forces transmitted by integrin molecules, are regulated by both mechanical and biochemical cues, and the outside-in and...

Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed. Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear. Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system. Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown). Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. Disclosures Zheng: Ablynx: Consultancy; Alexion: Research Funding.