IIb-IIIa Research Articles

New Insights into the Role of HMGB2 in ST-Segment Elevation Myocardial Infarction.

Ischemic heart disease is one of the leading causes of death in the world, of which ST-segment elevation myocardial infarction (STEMI) is an important type. Inappropriate activation and accumulation of platelets typically induced thrombosis, which may result in acute vessel occlusion and STEMI. Multiple cytokines have been shown to regulate platelet activation, but the relationship between HMGB2 and platelet activation has not been elucidated. We collected peripheral blood of STEMI patients and healthy adults, and mass spectrometry analysis of platelet proteins was conducted. The "edgeR" package was used to identify the differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene ontology (GO) and Gene Set Enrichment Analysis (GSEA) were used to identify the significantly changed pathways. Western blot and ELISA were used to detect the expression of a high mobility group box 2 (HMGB2). Flow cytometric analysis and platelet aggregation rate were performed to evaluate the activation of platelets. We identified ALOX5, HIST1H1B, S100A11, HMGB2, and RPS15A were the top five up-regulated proteins by differential expression analysis. Western blot verified that the relative protein expression of HMGB2 in platelet was significantly higher in STEMI patients compared with control adults, and the results of ELISA indicated that the serum HMGB2 level increased and significantly correlated with neutrophil count in STEMI patients. Further investigation showed that the platelet aggregation induced by ADP, the activation of integrin αIIbβ3 and CD62P expression on platelet surface were all enhanced by the recombinant HMGB2 (rHMGB2). In conclusion, HMGB2 may be the key molecule to regulate platelet activation in patients with STEMI, which may serve as a potential therapeutic target for STEMI.

Impact of age on pre-procedural TIMI flow in STEMI patients undergoing primary percutaneous coronary intervention.

Advanced age is a major determinant of impaired prognosis among patients with ST-segment elevation myocardial infarction (STEMI). However, the mechanisms associated with suboptimal reperfusion and enhanced complications are still largely undefined. The aim of the present study was to assess the impact of age on the angiographic findings and the procedural results of primary percutaneous coronary intervention (pPCI) in patients with STEMI. A consecutive cohort of patients admitted for STEMI treated with pPCI were included. Infarct-related artery (IRA) patency was defined for preprocedural TIMI flow 3. We included 520 patients, divided according to age tertiles (<61; 61-72; ≥73). Elderly patients were more often females, with hypertension, renal failure, prior myocardial infarction or PCI, with lower rates of smoking history, haemoglobin, leukocytes and cholesterol (P < 0.001), lower ejection fraction (P = 0.02), higher use of renin angiotensin system inhibitors, statins, ASA, calcium antagonists, diuretics and beta blockers. At angiography, for the IRA, percentage of thrombus (P = 0.02) and stenosis (P = 0.01), direct stenting (P = 0.02) and glycoprotein IIb-IIIa inhibitors (P = 0.04) inversely related with age, but for higher restenosis (P = 0.04). IRA patency was more common in patients aged ≥73 years (27.9% vs. 32.3% vs. 41.1%, P = 0.01). The impact of age on preprocedural TIMI flow was confirmed at multivariate analysis [adjusted odds ratio (95% confidence interval) = 0.68 (0.47-0.98), P = 0.04]. The present study shows that among STEMI patients undergoing primary PCI, more advanced age represents an independent predictor of preprocedural IRA patency. Future studies will define the implications on procedural results and long-term prognosis.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Regulation of Glycoprotein VI-Dependent Platelet Activation and Thrombus Formation by Heparan Sulfate Proteoglycan Perlecan.

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2β1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbβ3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

Biochemical and functional characteristics of stored (double-dose) buffy-coat platelet concentrates treated with amotosalen and a prototype UVA light-emitting diode illuminator.

Pathogen reduction of platelet concentrates (PCs) using amotosalen and broad-spectrum UVA illumination contributes to the safety of platelet transfusion by reducing the risk of transfusion-transmitted infections. We evaluated the in vitro quality of stored buffy-coat (BC) PCs treated with amotosalen and a prototype light-emitting diode (LED) illuminator. Double-dose BC-PCs collected into PAS-III/plasma or SSP+ /plasma (55/45%) were treated with amotosalen in combination with either conventional UVA lamps (INT100 Illuminator 320-400 nm) or LED illuminators at 350 nm. Platelet quality and function were evaluated over 7 days. Platelet counts were conserved during storage in all groups, as was platelet swirling without appearance of macroscopic aggregates. Integrin αIIbβ3 and glycoprotein (GP) VI expression remained stable, whereas GPIbα and GPV declined similarly in all groups. UV lamp- and LED-treated PCs displayed similar glucose consumption, lactate generation, and pH variation. Comparable spontaneous and residual P-selectin and phosphatidylserine exposure, activated αIIbβ3 exposure, mitochondrial membrane potential, lactate dehydrogenase release, and adhesive properties under flow conditions were observed during storage. The use of SSP+ /plasma compared with PAS-III/plasma better preserved most of these parameters, especially during late storage, irrespective of the type of illuminator. Replacing the UVA lamp for photochemical treatment by LED illuminators had no impact on platelet metabolism, spontaneous activation, apoptosis or viability, or on the in vitro function of BC-PCs stored for 7 days in SSP+ or PAS-III/plasma. These findings support improved procedures for the pathogen reduction and storage of PCs, to ensure transfusion safety and retention of platelet functional properties.

Clot Retraction and Its Correlation with the Function of Platelet Integrin αIIbβ3.

Clot retraction results from retractions of platelet filopodia and fibrin fibers and requires the functional platelet αIIbβ3 integrin. This assay is widely used to test the functions of platelets and fibrinogen as well as the efficacy of fibrinolysis. Changes in clot retraction have been found in a variety of hemostatic abnormalities and, more recently, in arterial thrombosis. Despite its broad clinical use and low cost, many aspects of clot retraction are poorly understood. In the present study, we performed two clinical standard clot retraction assays using whole-blood and platelet-rich plasma (PRP) samples to determine how clot retraction correlates with platelet counts and mean volume, the density of αIIbβ3 integrin and PLA genotypes, and plasma fibrinogen levels. We found that clot retraction was affected by platelet counts, but not mean platelet volume. It correlated with the surface density of the integrin αIibβ3, but not PLA genotypes. These results indicate that clot retraction measures a unique aspect of platelet function and can serve as an additional means to detect functional changes in platelets.

Shedding light into the biological activity of aminopterin, via molecular structural, docking, and molecular dynamics analyses

In this study, the structural and anticancer properties of aminopterin, as well as its antiviral characteristics, were elucidated. The preferred conformations of the title molecule were investigated with semiempirical AM1 method, and the obtained the lowest energy conformer was then optimized by using density functional (DFT/B3LYP) method with 6-311++G(d,p) as basis set. The vibrational frequencies of the optimized structure were calculated by the same level of theory and were compared with the experimental values. The vibrational assignments were performed based on the computed potential energy distribution (PED) of the vibrational modes. The molecular electrostatic potential (MEP) and frontier molecular orbitals (HOMO, LUMO) analyses were carried out for the optimized structure and the chemical reactivity has been scrutinized. To enlighten the biological activity of aminopterin as anticancer and anti-COVID-19 agents, aminopterin was docked into DNA, αIIBβ3 and α5β1integrins, human dihydrofolate reductase, main protease (Mpro) of SARS-CoV-2 and SARS-CoV-2/ACE2 complex receptor. The binding mechanisms of aminopterin with the receptors were clarified. The molecular docking results revealed the strong interaction of the aminopterin with DNA (−8.2 kcal/mol), αIIBβ3 and α5β1 integrins (−9.0 and −10.8 kcal/mol, respectively), human dihydrofolate reductase (−9.7 kcal/mol), Mpro of SARS-CoV-2 (−6.7 kcal/mol), and SARS-CoV-2/ACE2 complex receptor (−8.1 kcal/mol). Moreover, after molecular docking calculations, top-scoring ligand-receptor complexes of the aminopterin with SARS-CoV-2 enzymes (6M03 and 6M0J) were subjected to 50 ns all-atom MD simulations to investigate the ligand-receptor interactions in more detail, and to determine the binding free energies accurately. The predicted results indicate that the aminopterin may significantly inhibit SARS-CoV-2 infection. Thus, in this study, as both anticancer and anti-COVID-19 agents, the versatility of the biological activity of aminopterin was shown. Communicated by Ramaswamy H. Sarma

HTRS2023.P1.4 Characterization of the binding interaction between factor XII and integrin αIIbβ3

HTRS2023.P1.4 Characterization of the binding interaction between factor XII and integrin αIIbβ3

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.

Daidzein Inhibits Human Platelet Activation by Downregulating Thromboxane A2 Production and Granule Release, Regardless of COX-1 Activity.

Platelets play crucial roles in cardiovascular diseases (CVDs) by regulating hemostasis and blood coagulation at sites of blood vessel damage. Accumulating evidence indicates daidzein inhibits platelet activation, but the mechanism involved has not been elucidated. Thus, in this study, we investigated the mechanism responsible for the inhibition of collagen-induced platelet aggregation by daidzein. We found that in collagen-induced platelets, daidzein suppressed the production of thromboxane A2 (TXA2), a molecule involved in platelet activation and aggregation, by inhibiting the cytosolic phospholipase A2 (cPLA2) signaling pathway. However, daidzein did not affect cyclooxygenase-1 (COX-1). Furthermore, daidzein attenuated the PI3K/PDK1/Akt/GSK3αβ and MAPK (p38, ERK) signaling pathways, increased the phosphorylation of inositol trisphosphate receptor1 (IP3R1) and vasodilator-stimulated phosphoprotein (VASP), and increased the level of cyclic adenosine monophosphate (cAMP). These results suggest that daidzein inhibits granule release (ATP, serotonin, P-selectin), integrin αIIbβ3 activation, and clot retraction. Taken together, our study demonstrates that daidzein inhibits collagen-induced platelet aggregation and suggests that daidzein has therapeutic potential for the treatment of platelet aggregation-related diseases such as atherosclerosis and thrombosis.

Response of human blood platelets to preparations from leaves of Paulownia Clon in Vitro 112

Paulownia Clon in Vitro 112, also called the Oxytree, is a fast-growing hybrid of two trees belonging to the Paulowniaceae family – P. elongata and P. fortunei. It demonstrates a wide range of biological effects (including antioxidant, anti-inflammatory, antibacterial, and neuroprotective) due to the high concentration of secondary metabolites. Our previous results showed an in vitro antioxidant and antiplatelet activity of the extract and four fractions (A–D) from the leaves of Paulownia Clon in Vitro 112 in human plasma and washed blood platelets. Here, we used a microchip flow chamber-based thrombus formation analysis system (T-TAS) and flow cytometry to assess the anticoagulant and antiplatelet activity of the extract and four fractions with different chemical content (A-D) from Paulownia Clon in Vitro 112 leaves in human whole blood. Two tested fractions: fraction C and D (at the concentrations of 5 and 50 μg/mL) inhibited the exposition of the active form of GPIIb/IIIa (integrin αIIbβ3) on the surface of blood platelets stimulated by ADP and collagen. The antiplatelet activity of fraction C is likely due to its high verbascoside content and the presence of apigenin’s derivatives. Fraction D contains triterpenoids, including ursolic, pomoleic, and maslinic acid, which could be responsible for decreased activation of ADP- and collagen-stimulated blood platelets. These results suggest that fractions C and D might be promising sources of phytochemicals with antiplatelet activity, which are important for prophylaxis and treatment of cardiovascular diseases associated with hyperactivation of blood platelets. However, further research is needed to ascertain which exact compounds and mechanisms are responsible for this phenomenon.

Analysis of the epidemiological characteristics of posterior malleolus fracture in adults

BackgroundThis study explores the latest epidemiological characteristics of posterior malleolus fracture and compares the epidemiological differences of posterior malleolus fracture in different periods, regions, and adult age groups.MethodsClinical information of inpatients with posterior malleolus fracture in Shanghai Tongji Hospital and Karamay Central Hospital from January 2014 to February 2022 was reviewed and collected. The imaging data of patients were acquired using the Picture Archiving and Communication Systems. A statistical analysis was performed as to gender, current age, year of admission, injury mechanism, fracture type, and posterior malleolus fracture classification. Moreover, a comparative analysis was conducted on the injury mechanisms and morphological differences of posterior malleolus fracture at different periods, regions, and age groups.ResultsA total of 472 patients (210 patients from Shanghai Tongji Hospital and 262 patients from Karamay Central Hospital) with posterior malleolus fracture and an average age of 48.7 ± 15.6 were included in this study. The peak of posterior malleolus fracture occurs in the age group of 50–59. The injury mechanisms mainly involve low-energy fall and sprain (411 cases, 87.1%), followed by traffic accidents (52 cases, 11.0%), and fall injury from height (9 cases, 1.9%). With aging, the number of fall and sprain cases increases and reaches the peak at the age of 50–59, followed by progressive decline. Traffic accidents presents a relatively flat small peak in the age group of 40–59. The number of cases according to different fracture types shows the following ascending order: trimalleolar fracture-supination external rotation (335 cases, 71.0%) > bimalleolar fracture (60 cases, 12.7%) > trimalleolar fracture-pronation extorsion (43 cases, 9.1%) > posterior malleolus + tibial shaft fracture (19 cases, 4.0%) > simple posterior malleolus fracture (15 cases, 3.2%). The numbers of cases corresponding to the Haraguchi I Type, II Type, and III Type of posterior malleolus fractures were 369 (78.2%), 49 (10.4%), and 54 (11.4%), respectively. The Tongji IIA Type represented the highest number of cases (249 cases, 52.8%), followed by the IIB Type (120 cases, 25.4%), I Type (54 cases, 11.4%), IIIB Type (36 cases, 7.6%), and IIIA type (13 cases, 2.8%). The trimalleolar fracture-supination external rotation, Haraguchi I Type and Tongji IIA Type of posterior malleolus fractures all presented an obvious peak of incidence in the age group of 50–59. However, no obvious statistical difference was observed in the injury mechanism, Haraguchi classification, and Tongji classification of posterior malleolus fractures among different years and regions in recent years (P > 0.05).ConclusionsThe injury mechanism of posterior malleolus fracture mainly involves low-energy fall and sprain cases. The trimalleolar fracture-supination external rotation, Haraguchi I type and Tongji IIA type of posterior malleolus fracture are predilection fracture types, and all present an obvious incidence peak in the age group of 50–59. Elderly patients have high risks of falling and their bones are more fragile, conditions which are potential risk factors of posterior malleolus fracture. Early positive control has important significance. This study provides references for relevant basic and clinical studies of posterior malleolus fracture.

Use of Glycoprotein IIb-IIIa Inhibitors in Patients Undergoing Carotid Artery Stenting in the Vascular Quality Initiative

Antiplatelet therapies with thromboxane inhibitors and adenosine 5'-diphosphate antagonists have been widely used following carotid artery stenting (CAS). However, these therapies may not apply to patients who are intolerant or present acutely. Glycoprotein IIb/IIIa inhibitors (GPI) are a proposed alternative therapy in these patients; however, their use has been limited due to concerns of increased risk for intracranial bleeding. Thus, this study aims to assess the safety profile of GPI in patients undergoing CAS. All patients undergoing CAS in the Society of Vascular Surgery - Vascular Quality Initiative database from 2012 to 2021 was included and grouped into GPI versus non-GPI therapy (control). The primary outcome was in-hospital stroke or death, and secondary outcomes included in-hospital stroke/transient ischemic attack (TIA), death, myocardial infarction, and intracranial hemorrhage (ICH)/seizure. Patients were stratified by surgical approach (Transcarotid artery revascularization using flow reversal (TCAR) and transfemoral carotid artery stenting), and stepwise backward logistic regression analysis was conducted to evaluate major primary and secondary outcomes. A total of 50,628 patients underwent carotid revascularization. Of these, 4.4% of the patients received GPI. Mean age was similar between control versus GPI (71.35(9.67) vs. 71.36(10.20) years). Compared to the control group, patients who receive GPI are less likely to be on optimal medical therapy, including aspirin (83.0% vs. 88.1%), P2Y12 inhibitor (73.0% vs. 82.7%), and statin (82.3% vs. 86.0%) (All P<0.05). In addition, patients in the GPI group were more likely to undergo TCAR for carotid revascularization (52.2% vs. 48.4%) for emergent/urgent (29.4% vs. 16.8%) and symptomatic indications (55.5% vs. 49.7%) (All P<0.001). After stratifying by surgical approach, if patients underwent TFCAS and received a GPI, they were at increased odds of developing stroke/death (1.77(1.25-2.51)), death (odds ratio (OR) (95% CI): 1.67(1.07-2.61)), stroke/TIA (OR (95% confidence interval (CI)): 1.65(1.09-2.51)), and ICH/seizure (OR (95% CI): 2.13(1.23-3.68)) (All P<0.05). No difference was seen in outcomes between the 2 groups if undergoing TCAR. Patients who receive GPI were more likely to be symptomatic at presentation and less likely to be medically optimized before their carotid revascularization. Transfemoral access in patients receiving GPI was associated with increased odds of morbidity and mortality. However, this was not observed if undergoing TCAR. TCAR can be considered for its overall favorable results in high-risk patients who are not medically optimized.

Clinical analysis of distal radius core decompression for chronic wrist pain

To investigate the effectiveness of distal radius core decompression in the treatment of chronic wrist pain caused by various etiologies. A retrospective analysis was performed for the clinical data of 10 patients with chronic wrist pain treated with distal radial core decompression between January 2018 and December 2021. There were 6 males and 4 females with an average age of 37.4 years (range, 21-55 years). The disease duration ranged from 7 to 72 months, with an average of 26.5 months. Preoperative MRI examination showed that 10 cases had bone marrow edema at the distal radius on the affected side, and 8 cases had bone marrow edema in the carpal bones such as scaphoid and lunate bone. Among them, 3 patients had a history of wrist fracture, and 2 patients had Kienböck diseases (1 case each in stage ⅡB and stage ⅢA). Three cases were combined with triangular fibrocartilage complex (TFCC) type 1A injury. Two cases were combined with osteoarthritis, 1 of them was complicated with severe traumatic arthritis, the wrist arthroscopy showed that the TFCC was completely lost and could not be repaired, and the cartilage of the lunate bone and the ulnar head were severely worn.Visual analogue scale (VAS) score was used to evaluate the relief of wrist pain before operation, at 6 months after operation, and at last follow-up, and the range of motion of the affected wrist in dorsiflexion, palmar flexion, ulnar deviation, and radial deviation was measured. The degree of bone marrow edema was evaluated according to T1WI, T2WI, and STIR sequences of MRI. All the patients were followed up 12-22 months, with an average of 16.4 months. Except for 1 patient who experienced persistent wrist joint pain and limited mobility after operation, the remaining 9 patients showed significant improvement in pain symptoms and wrist joint mobility. The VAS score and range of motion of wrist dorsiflexion, palmar flexion, ulnar deviation, and radial deviation at 6 months after operation and at last follow-up were significantly improved when compared with those before operation, the VAS score and the range of motion of wrist ulnar deviation and radial deviation at last follow-up were further improved when compared with those at 6 months after operation, all showing significant differences ( P<0.05). There was no significant difference in wrist dorsiflexion and palmar flexion between at 6 months after operation and at last follow-up ( P>0.05). Bone marrow edema was improved in 6 patients on MRI at 6 months after operation, and was also improved in other patients at last follow-up. For chronic wrist pain caused by a variety of causes, distal radius core decompression can directly reduce the pressure of the medullary cavity of the distal radius, improve the blood supply of the corresponding distal structure, significantly alleviate chronic wrist pain, and provide an option for clinical treatment.

Effects of Antiplatelet Drugs on Platelet-Dependent Coagulation Reactions.

Activated platelets are involved in blood coagulation by exposing phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes. Platelets accelerate fibrin formation and thrombin generation, two final reactions of the coagulation cascade. We investigated the effects of antiplatelet drugs on platelet impact in these reactions and platelet ability to expose PS. Washed human platelets were incubated with acetylsalicylic acid (ASA), ticagrelor, ASA in combination with ticagrelor, ruciromab (glycoprotein IIb-IIIa antagonist), or prostaglandin E1 (PGE1). Platelets were not activated or activated by collagen and sedimented in multiwell plates, and plasma was added after supernatant removal. Fibrin formation (clotting) was monitored in a recalcification assay by light absorbance and thrombin generation in a fluorogenic test. PS exposure was assessed by annexin V staining using flow cytometry. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, prolonged the lag phase and decreased the maximum rate of plasma clotting and decreased the peak and maximum rate of thrombin generation. Inhibition was observed when platelets were not treated with exogenous agonists (activation by endogenous thrombin) and pretreated with collagen. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, decreased PS exposure on washed platelets activated by thrombin and by thrombin + collagen. PS exposure on activated platelets in whole blood was lower in patients with acute coronary syndrome receiving ticagrelor + ASA in comparison with donors free of medications. These results indicate that antiplatelet drugs are able to suppress platelet coagulation activity not only in vitro but also after administration to patients.

Cryo-EM structures of full-length integrin αIIbβ3 in native lipids

Platelet integrin αIIbβ3 is maintained in a bent inactive state (low affinity to physiologic ligand), but can rapidly switch to a ligand-competent (high-affinity) state in response to intracellular signals (“inside-out” activation). Once bound, ligands drive proadhesive “outside-in” signaling. Anti-αIIbβ3 drugs like eptifibatide can engage the inactive integrin directly, inhibiting thrombosis but inadvertently impairing αIIbβ3 hemostatic functions. Bidirectional αIIbβ3 signaling is mediated by reorganization of the associated αIIb and β3 transmembrane α-helices, but the underlying changes remain poorly defined absent the structure of the full-length receptor. We now report the cryo-EM structures of full-length αIIbβ3 in its apo and eptifibatide-bound states in native cell-membrane nanoparticles at near-atomic resolution. The apo form adopts the bent inactive state but with separated transmembrane α-helices, and a fully accessible ligand-binding site that challenges the model that this site is occluded by the plasma membrane. Bound eptifibatide triggers dramatic conformational changes that may account for impaired hemostasis. These results advance our understanding of integrin structure and function and may guide development of safer inhibitors.

A single F153Sβ3 mutation causes constitutive integrin αIIbβ3 activation in a variant form of Glanzmann thrombasthenia.

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate exvivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate exvivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbβ3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report β3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sβ3 substitution within the βI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153β3. F153 is completely conserved among β3 of several species and all human β-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sβ3 also displays reduced levels of a constitutively activated αIIb-S153β3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153β3 is critical for maintaining the resting conformation of α2- and α1-helices of the βI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the βI-domain toward the constitutively active αIIbβ3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbβ3 activation. The data collectively demonstrate that disruption of F153β3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153β3 may be compensated by a hyperactive conformation that promotes viable hemostasis.

The WASH-complex subunit Strumpellin regulates integrin αIIbβ3 trafficking in murine platelets

The platelet specific integrin αIIbβ3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbβ3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbβ3 is increased by the translocation of internal granule pools to the plasma membrane. The WASH complex is the major endosomal actin polymerization-promoting complex and has been implicated in the generation of actin networks involved in endocytic trafficking of integrins in other cell types. The role of the WASH complex and its subunit Strumpellin in platelet function is still unknown. Here, we report that Strumpellin-deficient murine platelets display an approximately 20% reduction in integrin αIIbβ3 surface expression. While exposure of the internal αIIbβ3 pool after platelet activation was unaffected, the uptake of the αIIbβ3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbβ3-positive vesicular structures revealed an enrichment of protein markers, which are associated with the endoplasmic reticulum, Golgi complex and early endosomes in Strumpellin-deficient platelets. These results point to a so far unidentified role of the WASH complex subunit Strumpellin in integrin αIIbβ3 trafficking in murine platelets.

Mechanisms of glabridin inhibition of integrin αIIbβ3 inside-out signals and NF-κB activation in human platelets

BackgroundPlatelets play a crucial role in cardiovascular diseases (CVDs) and are activated by endogenous agonists like collagen. These agonists initiate signal transduction through specific platelet receptors, resulting in platelet aggregation. Glabridin, a prenylated isoflavonoid found in licorice root, is known for its significance in metabolic abnormalities. Glabridin has been observed to inhibit collagen-induced platelet aggregation, but the precise mechanisms, specifically concerning NF-κB activation and integrin αIIbβ3 signaling, are not yet fully understood.MethodsIn this study, platelet suspensions were prepared from healthy human blood donors, and the aggregation ability was observed using a lumi-aggregometer. The inhibitory mechanisms of glabridin in human platelets were evaluated through immunoblotting and confocal microscopy. The anti-thrombotic effects of glabridin were assessed by histological analysis of lung sections in acute pulmonary thromboembolism and by examining fluorescein-induced platelet plug formation in mesenteric microvessels in mice.ResultsGlabridin inhibited integrin αIIbβ3 inside-out signals such as Lyn, Fyn, Syk, and integrin β3 activation and NF-κB-mediated signal events, with similar potency to classical inhibitors BAY11-7082 and Ro106-9920. Glabridin and BAY11-7082 inhibited IKK, IκBα, and p65 phosphorylation and reversed IκBα degradation, while Ro106-9920 only reduced p65 phosphorylation and reversed IκBα degradation. BAY11-7082 reduced Lyn, Fyn, Syk, integrin β3, phospholipase Cγ2 and protein kinase C activation. Glabridin reduced platelet plug formation in mesenteric microvessels and occluded vessels in thromboembolic lungs of mice.ConclusionOur study revealed a new pathway for activating integrin αIIbβ3 inside-out signals and NF-κB, which contributes to the antiplatelet aggregation effect of glabridin. Glabridin could be a valuable prophylactic or clinical treatment option for CVDs.

Comparative study of interaction energies between αIIbβ3 integrin and the peptidic, peptidomimetic and non-peptidic ligands by quantum mechanics FMO-PIEDA calculations

Integrins belong to a family of cell adhesion receptors. To better understand an adhesion mechanism of integrins, fragmented molecular orbital (FMO) method with pair interaction energy decomposition analysis (PIEDA) was applied for integrin:ligand complexes. Interaction energies were evaluated between the amino acid residues including Mg2+ and Ca2+ ions at ligand-binding site of αIIbβ3 integrin and two peptide chains with the Ala-Gly-Asp (AGD)- and the Arg-Gly-Asp (RGD)-binding motifs, a cyclic peptide (eptifibatide), peptidomimetic ligands (tirofiban and L-739758) and poly(l-lactic acid) chain (PLA). The results indicate that Mg2+ and Ca2+ ions together with Asp224A, Asn215B, Asp159A and Lys125B of αIIbβ3 are the most important residues for a binding of the peptidic ligands while for the peptidomimetic ligands and PLA, interactions with Ca2+ ions are less significant than those with amino acid residues of αIIbβ3. For all complexes, a dominant part of interaction energy comes from electrostatic interactions. New developed antagonists of αIIbβ3 should mimic not only the interactions of the RGD motif but also the interactions of the backbone of a longer peptidic sequence (RGDV or AGDV) with the focus on the interactions of the antagonists with the ADMIDAS Ca2+ ion. An interaction pattern predicted for PLA was compared with the native peptidic ligands.