Lymphocytes, Mean Platelet Volume, and Albumin in Critically Ill COVID-19 Patients with Venous Thromboembolism.

As one of the frequent complications leading to poor prognosis in hospitalized COVID-19 patients, a better understanding of venous thromboembolism (VTE) in COVID-19 patients is needed. We conducted a single-center, retrospective study on 96 COVID-19 patients admitted to the intensive care unit (ICU) from April to June 2022, in Shanghai Renji Hospital. Records of these COVID-19 patients upon admission were reviewed for demographic information, co-morbidities, vaccinations, treatment, and laboratory tests. VTE occurred in 11 (11.5%) cases among 96 COVID-19 patients despite the standard thromboprophylaxis since ICU admission. In COVID-VTE patients, a significant increase in B cells and a decrease in Ts cells were observed and a strong negative correlation (r = -0.9524, P = .0003) was found between these two populations. In COVID-19 patients with VTE, increased MPV and decreased albumin levels were seen in addition to the common VTE indicators of D-dimer abnormalities. The altered lymphocyte composition in COVID-VTE patients is noteworthy. In addition to D-dimer, MPV and albumin levels might be novel indicators for the risk of VTE in COVID-19 patients.

Targeting platelet-derived CXCL12 impedes arterial thrombosis.

The prevention and treatment of arterial thrombosis continue to be clinically challenging, and understanding the relevant molecular mechanisms in detail may facilitate the quest to identify novel targets and therapeutic approaches that improve protection from ischemic and bleeding events. The chemokine CXCL12 augments collagen-induced platelet aggregation by activating its receptor CXCR4. Here we show that inhibition of CXCR4 attenuates platelet aggregation induced by collagen or human plaque homogenate under static and arterial flow conditions by antagonizing the action of platelet-secreted CXCL12. We further show that platelet-specific CXCL12 deficiency in mice limits arterial thrombosis by affecting thrombus growth and stability without increasing tail bleeding time. Accordingly, neointimal lesion formation after carotid artery injury was attenuated in these mice. Mechanistically, CXCL12 activated via CXCR4 a signaling cascade involving Bruton's tyrosine kinase (Btk) that led to integrin αIIbß3 activation, platelet aggregation, and granule release. The heterodimeric interaction between CXCL12 and CCL5 can inhibit CXCL12-mediated effects as mimicked by CCL5-derived peptides such as [VREY]4. An improved variant of this peptide, i[VREY]4, binds to CXCL12 in a complex with CXCR4 on the surface of activated platelets, thereby inhibiting Btk activation and preventing platelet CXCL12-dependent arterial thrombosis. In contrast to standard antiplatelet therapies such as aspirin or P2Y12 inhibition, i[VREY]4 reduced CXCL12-induced platelet aggregation and yet did not prolong in vitro bleeding time. We provide evidence that platelet-derived CXCL12 is involved in arterial thrombosis and can be specifically targeted by peptides that harbor potential therapeutic value against atherothrombosis.

Effects of the Btk-Inhibitors Remibrutinib (LOU064) and Rilzabrutinib (PRN1008) With Varying Btk Selectivity Over Tec on Platelet Aggregation and in vitro Bleeding Time.

Background: Bruton tyrosine kinase inhibitors (BTKi) are used in B-cell malignancies and in development against various autoimmune diseases. Since Btk is also involved in specific pathways of platelet activation, BTKi might be considered to target platelet GPVI/GPIb-mediated atherothrombosis and platelet FcγRIIA-dependent immune disorders. However, BTKi treatment of patients with B-cell malignancies is frequently associated with mild bleeding events caused possibly by off-target inhibition of Tec. Here, we compared the platelet effects of two novel BTKi that exhibit a high (remibrutinib) or low (rilzabrutinib) selectivity for Btk over Tec. Methods and Results: Remibrutinib and rilzabrutinib were pre-incubated with anticoagulated blood. Platelet aggregation and in vitro bleeding time (closure time) were studied by multiple electrode aggregometry (MEA) and platelet-function analyzer-200 (PFA-200), respectively. Both BTKi inhibited atherosclerotic plaque-stimulated GPVI-mediated platelet aggregation, remibrutinib being more potent (IC50 = 0.03 µM) than rilzabrutinib (IC50 = 0.16 µM). Concentrations of remibrutinib (0.1 µM) and rilzabrutinib (0.5 µM), >80% inhibitory for plaque-induced aggregation, also significantly suppressed (>90%) the Btk-dependent pathways of platelet aggregation upon GPVI, von Willebrand factor/GPIb and FcγRIIA activation stimulated by low collagen concentrations, ristocetin and antibody cross-linking, respectively. Both BTKi did not inhibit aggregation stimulated by ADP, TRAP-6 or arachidonic acid. Remibrutinib (0.1 µM) only slightly prolonged closure time and significantly less than rilzabrutinib (0.5 µM). Conclusion: Remibrutinib and rilzabrutinib inhibit Btk-dependent pathways of platelet aggregation upon GPVI, VWF/GPIb, and FcγRIIA activation. Remibrutinib being more potent and showing a better profile of inhibition of Btk-dependent platelet activation vs. hemostatic impairment than rilzabrutinib may be considered for further development as an antiplatelet drug.

Spontaneous Platelet Aggregation in Blood Is Mediated by FcγRIIA Stimulation of Bruton's Tyrosine Kinase.

High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2-3 min. In our cohort of healthy blood donors (n = 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.

Chemokines and galectins form heterodimers to modulate inflammation.

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin-1 and galectin-3, we identified several interacting pairs, such as CXCL12 and galectin-3. Based on NMR and MD studies of the CXCL12/galectin-3 heterodimer, we identified contact sites between CXCL12 ß-strand 1 and Gal-3 F-face residues. Mutagenesis of galectin-3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin-3, but not its mutants, inhibited CXCL12-induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin-3 attenuated CXCL12-stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.

Oral Bruton tyrosine kinase inhibitors block activation of the platelet Fc receptor CD32a (FcγRIIA): a new option in HIT?

Activation of the platelet Fc-receptor CD32a (FcγRIIA) is an early and crucial step in the pathogenesis of heparin-induced thrombocytopenia type II (HIT) that has not been therapeutically targeted. Downstream FcγRIIA Bruton tyrosine kinase (BTK) is activated; however, its role in Fc receptor-induced platelet activation is unknown. We explored the potential to prevent FcγRIIA-induced platelet activation by BTK inhibitors (BTKi's) approved (ibrutinib, acalabrutinib) or in clinical trials (zanubrutinib [BGB-3111] and tirabrutinib [ONO/GS-4059]) for B-cell malignancies, or in trials for autoimmune diseases (evobrutinib, fenebrutinib [GDC-0853]). We found that all BTKi's blocked platelet activation in blood after FcγRIIA stimulation by antibody-mediated cross-linking (inducing platelet aggregation and secretion) or anti-CD9 antibody (inducing platelet aggregation only). The concentrations that inhibit 50% (IC50) of FcγRIIA cross-linking-induced platelet aggregation were for the irreversible BTKi's ibrutinib 0.08 µM, zanubrutinib 0.11 µM, acalabrutinib 0.38 µM, tirabrutinib 0.42 µM, evobrutinib 1.13 µM, and for the reversible BTKi fenebrutinib 0.011 µM. IC50 values for ibrutinib and acalabrutinib were four- to fivefold lower than the drug plasma concentrations in patients treated for B-cell malignancies. The BTKi's also suppressed adenosine triphosphate secretion, P-selectin expression, and platelet-neutrophil complex formation after FcγRIIA cross-linking. Moreover, platelet aggregation in donor blood stimulated by sera from HIT patients was blocked by BTKi's. A single oral intake of ibrutinib (280 mg) was sufficient for a rapid and sustained suppression of platelet FcγRIIA activation. Platelet aggregation by adenosine 5'-diphosphate, arachidonic acid, or thrombin receptor-activating peptide was not inhibited. Thus, irreversible and reversible BTKi's potently inhibit platelet activation by FcγRIIA in blood. This new rationale deserves testing in patients with HIT.

Downregulation of Pin1 in human atherosclerosis and its association with vascular smooth muscle cell senescence.

OBJECTIVE: Pin1 is prevalently overexpressed in human cancers and implicated to regulate cell growth and apoptosis. Thus far, however, no role for Pin1 has been described in modulating vascular smooth muscle cell (VSMC) senescence. METHODS: Immunohistochemistry and Western blotting were used to assess Pin1 protein level in human normal and atherosclerotic tissues. ß-galactosidase staining, cumulative population doubling level, telomerase activity, and relative telomere length measurement were used to confirm VSMC senescence. The expressions of Pin1 and other genes involved in this research were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blotting in VSMCs. Apolipoprotein E gene-deleted mice (ApoE-/-) fed a high-fat diet were treated with juglone or 10% ethanol, respectively, for 3 weeks. The extent of atherosclerosis was evaluated by Oil Red O, Masson trichrome staining, and immunohistology. RESULTS: Pin1 protein level decreased in human atherosclerotic tissues and VSMCs, synchronously with increased VSMC senescence. Adenoviral-mediated Pin1 overexpression rescued cellular senescence in atherosclerotic VSMCs, with concurrent down-regulation of P53, p21, growth arrest and DNA-damage-inducible protein 45-alpha (Gadd45a), phosphorylated retinoblastoma (p-pRb), p65 and upregulation of cyclin subfamilies (cyclin B, D, and E), and cyclin-dependent kinase subfamilies (2, 4, and 6), whereas Pin1 knockdown resulted in the converse effects, indicating that VSMC senescence mediated by Pin1 is an integrated response to diverse signals. In vivo data from ApoE-/- mice showed that treatment of juglone led to accelerated atherosclerosis development. CONCLUSIONS: Altogether this work supports a role for Pin1 as a vital modulator of VSMC senescence, thereby providing a novel target for regulation and control of atherosclerosis.

Prevention of Neointimal Hyperplasia by Local Application of Lentiviral Vectors Encoding Pin1 shRNA in Pluronic F127.

Inhibition of intimal hyperplasia plays an important role in preventing restenosis. Previously, we reported the provocative role of Pin1 in regulating vascular smooth muscle cell (VSMC) proliferation. Here we intended to identify whether locally delivered lentivirus-mediated siPin1 via pluronic F127 (PF127) could inhibit neointimal formation and further explore the potential mechanisms thereof. In vitro studies revealed that lentivirus-mediated siPin1 dispersed in PF127 suppressed proliferation and induced senescence in VSMCs. Reduction of Pin1 expression resulted in a decrease of phospho-Akt (p-Akt) expression level in VSMCs. Reactivation of Akt phosphorylation overcame the siPin1-mediated senescence. In a rat wire injury model, periadventitial delivery of lentivirus-mediated siPin1 via PF127 produced inhibition of intimal hyperplasia 14 days after injury without evidence for toxicity. Furthermore, the reduction of intimal thickness was associated with a decreased amount of PCNA positive cells, decreased telomerase activity and shortened telomere length. Therefore, these results suggest that PF127 delivery of lentivirus-mediated siPin1 to artery may have a therapeutic potential for the treatment of restenosis.