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The rare but dangerous adverse events evidenced after massive vaccination against SARS-CoV-2 are represented by thrombosis and thrombocytopenia. The patients diagnosed with severe COVID-19 may develop a pro-thrombotic state with a much higher frequency, thus we decided to investigate the role of Spike protein (the only common product of the two conditions) or the anti-Spike antibodies in the etiopathogenesis of thrombosis. A pathogenic Platelet Factor 4 (PF4)-dependent syndrome, unrelated to the use of heparin therapy, has been reported after the administration of vaccines in the patients manifesting acute thrombocytopenia and thrombosis. Thus, we aimed at shedding light on the structural similarities of Spike of SARS-CoV-2 and PF4 on their eventual biochemical interactions and on the role of their specific antibodies. The similarities between PF4 and Spike-RBD proteins were evaluated by a comparison of the structures and by testing the cross-reactivity of their specific antibodies by ELISA assays. We found that the anti-Spike antibodies do not recognize PF4, on the contrary, the anti-PF4 antibodies show some cross-reactivity for Spike-RBD. More interestingly, we report for the first time that the PF4 and Spike-RBD proteins can bind each other. These data suggest that the interaction of the two proteins could be involved in the generation of anti-PF4 antibodies, their binding to Spike-RBD, which could lead to platelets aggregation due also to their high expression of ACE2. [ABSTRACT FROM AUTHOR]

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: The development of chemically defined media has become a particularly important task for in vitro embryo production systems, which require maintained reproducible results when new additives are tested for culture, beyond observational studies. Specifically, we need studies measuring the impact of these media on the embryonic transcriptome, particularly those negatively affecting embryo quality. Consequently, this study evaluated by using a microarray approach the transcriptome of porcine embryos produced in vitro, cultured in a defined vs. an undefined medium and contrasted with in vivo-derived embryos. No significantly altered genes were found between in vitro-produced embryos, despite the theoretical limitations that usually accompany defined media. However, when they were compared with in vivo-derived embryos, many altered genes were observed, reflecting how current culture conditions deeply modify the embryonic transcriptome. A better understanding of these alterations may offer new ways to improve in vitro embryo production systems. Likewise, developing a chemically defined medium capable of producing embryos of a similar quality to traditional media may contribute to this task. The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA. [ABSTRACT FROM AUTHOR]

Vaccine-induced immune thrombotic thrombocytopenia (VITT) (also termed thrombosis with thrombocytopenia syndrome or vaccine-induced thrombotic thrombocytopenia or vaccine-induced immune thrombocytopenia) is characterized by (i) venous or arterial thrombosis; (ii) mild-to-severe thrombocytopenia; (iii) positive antiplatelet factor 4 (PF4)-polyanion antibodies or anti-PF4-heparin antibodies detected by the HIT (heparin-induced thrombocytopenia) ELISA; (iv) occurring 5-30 days after ChAdOx1 nCoV-19 (AstraZeneca) or Ad26.COV2.S (Johnson & Johnson/Janssen) vaccination. VITT's incidence is 1 per 100 000 vaccinated people irrespective of age and up to 1 in 50 000 for people <50 years of age with the AstraZeneca COVID-19 vaccine. The exact mechanism by which adenovirus-vectored COVID-19 vaccines trigger this syndrome is still unclear, as for the increased risk for acute cerebral sinus venous thrombosis and splanchnic vein thrombosis as compared to other locations of venous thrombotic events. VITT is associated with the detection of anti-PF4 antibodies, unrelated to previous use of heparin therapy. PF4 antibodies are thought to activate platelets via the platelet FcγRIIA receptors leading to further platelet activation that causes thrombosis and thrombocytopenia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Abstract: Preparation for storage may cause platelet activation. The quality of platelet concentrates plays an important role in transfusion therapy. Platelet concentrates are produced by different centrifugation methods; buffy coat (buffy coat-derived platelet concentrates-BC) and plateletpheresis (apheresis-derived platelet concentrates-APC). Their quality was assessed using the following parameters: platelet, WBC and RBC counts pH, volume, platelet factor 4 (PF4) and Annexin V. The present paper compares the quality of both platelet preparations in vitro. In this experimental study, 30 platelet concentrates were harvested with the Haemonetics MCS plus and 30 units via the buffy coat (BC) method. The percentages of Annexin V expression, PF4 levels, platelet, WBC and RBC counts, pH and volume were measure immediately after collection and after 3days of storage. During storage for up to 3days, BC units displayed, no significant pH or RBC, difference in comparison with apheresis preparations (p >0.05). During storage for up to 3days, BC units displayed a significant increase in the PF4 and Annexin V expression, compared to the apheresis preparations on day three (p <0.05). The kinetics of PF4 and Annexin V levels are influenced by the method used to prepare platelets for storage. The different levels of PF4 and Annexin V in BCs and APCs clearly demonstrates a progressive activation of BC platelets exceeding that of APC. However, in vivo studies should be performed to confirm these findings. [Copyright &y& Elsevier]

Platelet factor 4 (PF4, CXCL4) is a small chemokine protein released by activated platelets. Although a major physiological function of PF4 is to promote blood coagulation, this cytokine is involved in innate and adaptive immunity in events when platelets are activated in response to infections. Coronavirus disease 2019 (COVID-19) patients have abnormal coagulation activities, and severe patients develop higher D-dimer levels. D-dimers are small protein products present in the blood after blood clots are degraded by fibrinolysis. To prevent clotting, heparin is often clinically used in COVID-19 patients. Some clinical procedures for the management of COVID-19 patients may include extracorporeal membrane oxygenation (ECMO) and renal replacement therapy (CRRT), which also require the use of heparin. Anti-PF4 antibodies are frequently detected in severe patients and heparin-induced thrombocytopenia (HIT) can also be observed. PF4 and its role in HIT as well as in pathologies seen in COVID-19 patients define a potential therapeutic option of using blocking antibodies in the treatment of COVID-19. [ABSTRACT FROM AUTHOR]

Retinal neovascularization (RNV) is a common pathology of blinding proliferative retinopathies. The current treatments to RNV, however, are hindered by limited efficacy, side effects, and drug resistance. A naturally-occurring cytokine in retina that is amicable to immune system and possesses robust anti-neovascular function would facilitate to overcome the hurdles. In this study, retinas from a mouse model of oxygen-induced retinopathy (OIR) underwent a protein array to screen the naturally-occurring cytokines that may antagonize RNV. Among the 62 angiogenesis-associated cytokines, platelet factor 4 (Pf4) stood out with the most prominent upregulation and statistical significance. Moreover, an intravitreal injection of mouse Pf4 demonstrated dramatic anti-vaso-obliteration and anti-neovascularization effects dose dependently in the OIR model; whereas human PF4 inhibited the proliferation, migration, and tubulogenesis of monkey retinal vascular endothelial cells treated with VEGF and TNF-α. These previously undescribed angiostatic effects of PF4 in OIR retinas and retinal vascular endothelial cells support translation of this naturally-occurring chemokine into a therapeutic modality to RNV supplementary to the anti-VEGFs. Mechanistically, a phosphorylation array and western blots indicated that downregulation of proline-rich Akt substrate of 40 kDa (Pras40) and its phosphorylation were necessary for Pf4's anti-neovascular effects in the OIR retinas. Indeed, overexpression of the wildtype Pras40 and the mutant version with deficient phosphorylation abolished and mimicked the Pf4's angiostatic effects in the OIR retinas, respectively. The similar effects were also observed in vitro. This study, for the first time, links PF4's anti-RNV function to an intracellular signaling molecule PRAS40 and its phosphorylation. • Previously undescribed anti-neovascularization and anti-vaso-obliteration of Pf4 in retina of OIR mouse model. • Profound inhibition of PF4 on pathological angiogenesis in retinal vascular endothelial cells. • PF4's angiostatic effects and upregulation in PDR patients support its translation into a novel therapeutic to RNV. • PF4's anti-RNV is, for the first time, linked to an intracellular signaling molecule PRAS40 and its phosphorylation. [ABSTRACT FROM AUTHOR]

Due to the variable overlap of multiple symptoms, accurate early diagnosis of NK/T-cell lymphoma-associated hemophagocytic syndrome (NK/T-LAHS) is difficult, making the prognosis extremely poor. Hemophagocytic syndrome (HPS) is now diagnosed primarily based on the hemophagocytic lymphohistiocytosis (HLH)-2004 diagnostic criteria, and platelet count is one of the baseline evaluations. However, in our study, the data showed that decreased platelets were not only a clinical feature of HPS but also the key cells that regulate inflammation by releasing α-granules containing upregulated platelet factor 4 (PF4) and downregulated platelet-derived growth factors (PDGFs). Furthermore, we found that angiopoietin-4 (ANG-4), which has significant differential expression, has been less reported, that may affect hematopoiesis and proinflammatory responses and can be used as diagnostic biomarkers together with PF4 and PDGFs. [ABSTRACT FROM AUTHOR]

Metastasis of melanoma to the distant organs is a multistep process in which the tumor microenvironment (TME) may play an important role. However, the relationship between metastatic progression and TME is intricate. In the present study, using melanoma derivative cell lines OL (oligometastatic) and POL (polymetastatic) that differ in their metastatic colonization capability, we have elucidated a new mechanism involving "SEC23A-PF4-MAPK/ERK axis" in which PF4 transported by COPII hinders metastasis through inhibition of MAPK/ERK signaling pathway. Furthermore, SPARC can act cooperatively to enhance the inhibition of Pf4 on ERK phosphorylation and melanoma cell metastasis. Our findings show the possibility of targeting cancer cell secretome for therapeutic development., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo-epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram-negative Escherichia coli., Objectives: To assess the role of PF4, anti-PF4/H antibodies and FcγRIIa in killing of Gram-positive bacteria by platelets., Methods: Binding of PF4 to protein-A deficient Staphylococcus aureus (SA113Δspa) and non-encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti-PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P-expression, PAC-1 binding) and phosphatidylserine (PS)-exposure were analyzed., Results: Monoclonal and patient-derived anti-PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6-fold increased fluorescence signal for human anti-PF4/H antibodies to 24.0-fold increase for KKO). Staphylococcus aureus (5.5 × 10 4 cfu/mL) was efficiently killed by platelets (2.7 × 10 4 cfu/mL) or their releasate (2.9 × 10 4 cfu/mL). Killing was not further enhanced by PF4 or anti-PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP-6 stimulation and exposed high levels of PS., Conclusions: Anti-PF4/H antibodies seem to have only a minor role for direct killing of Gram-positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Heparin-induced thrombocytopenia (HIT), even rare, is a life-threatening, immune-mediated complication of heparin exposure. It is considered the most severe non-bleeding adverse reaction of heparin treatment and one of the most important adverse drug reactions. The pathophysiological basis of HIT results from the formation of an immunocomplex consisting of an auto-antibody against platelet factor 4 (PF4) - heparin complex, which binds to the surface of platelets and monocytes, provoking their activation by cross-linking FcgIIA receptors. Platelets and monocyte activation, leads to the generation of catastrophic arterial and venous thrombosis, with a mortality rate of 20%, without early recognition. The definitive diagnosis of HIT i.e., clinical and laboratory evidence, can not be done at the onset of symptoms because laboratory results may not be available for several days. Thus, the initial approach is to predict the likelihood of HIT, because in highly suspected patients immediate heparin cessation and initiation of alternative anticoagulation treatment are crucial for the prevention of the devastating thrombotic sequelae. Herein, we describe the pathophysiology, the clinical manifestations, the diagnostic approach, and the management of patients with HIT., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Patriarcheas et al.)

Hematological abnormalities after transplantation are complications that may arise after renal transplantation, of which thrombocytopenia is associated with increased risk of bleeding and other complications. The development of thrombocytopenia is affected by various clinical conditions, and the stromal-derived factor 1 (SDF1) and platelet factor 4 (PF4) genes are known to be involved in the production or destruction of platelets. The purpose of this study was to investigate the prevalence of posttransplant thrombocytopenia and its association with other clinical conditions and genetic polymorphisms of SDF1 and PF4 genes a long time after transplantation. This is a retrospective study that includes a total of 305 kidney transplant (KT) recipients between 2008 and 2012 at St. Vincent Medical Center, Los Angeles, CA. In this study, posttransplant thrombocytopenia was defined as a 30% reduction in platelet count from the baseline in the first week or a decrease of <100 (×103/μL) within 1 year after KT. The subjects were divided into posttransplant thrombocytopenia and control groups. The chi-square test, t- test, and logistic regression were used for the analyses. In the first week, 65 patients had a 30% reduction in platelet count (21.3%). Gender, simultaneous kidney-pancreas transplantation, induction therapy (IT), and only alleles of rs2297630 of SDF1, among the SDF1 and PF4 genes, showed statistically significant differences. The rs2297630 alleles were consistently significant risk factors (non G vs. G: odds ratio = 0.445; 95% confidence interval, 0.224–0.884; p =.021) in the multiple logistic regression. In the 1-year study, 61 patients (20.0%) had platelet counts of <100 × 103/μL and had statistically significant differences in patients who had delayed graft function and induction therapy. In this study, non-G group of rs2297630 in SDF1 significantly increased the risk of post-transplant thrombocytopenia in the first week of kidney transplantation. • PLT counts mostly decreased in the 1 week after KT. • The prevalence of PTT is related to gender, simultaneous kidney-pancreas transplantation, induction therapy (IT), and only genotype of rs2297630 of SFD1 in 1 week after KT. • The prevalence of PTT is related to delayed graft function and IT within 1 year after KT. • Non-G group of rs2297630 among SDF1 and PF4 genetic polymorphism is affected the PTT in 1 week but not within 1 year after KT. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4) is an anti- Plasmodium component of platelets. It is expressed in megakaryocytes and released from platelets following infection with Plasmodium Innate immunity is crucial for the host anti- Plasmodium response, in which type I interferon plays an important role. Whether there is cross-talk between innate immune signaling and the production of anti- Plasmodium defense peptides is unknown. Here we demonstrate that E74, like ETS transcription factor 4 (ELF4), a type I interferon activator, can help protect the host from Plasmodium yoelii infection. Mechanically, ELF4 binds to the promoter of genes of two C-X-C chemokines, Pf4 and pro-platelet basic protein ( Ppbp ), initiating the transcription of these two genes, thereby enhancing PF4-mediated killing of parasites from infected erythrocytes. Elf4 -/- mice are much more susceptible to Plasmodium infection than WT littermates. The expression level of Pf4 and Ppbp in megakaryocytes from Elf4 -/- mice is much lower than in those from control animals, resulting in increased parasitemia. In conclusion, our study uncovered a distinct role of ELF4, an innate immune molecule, in host defense against malaria., (© 2019 Wang et al.)

Background: The precise mechanisms of the increased incidence of hemostatic abnormalities in congenital heart disease (CHD) have not been determined. The aim of the study was to evaluate some indicators of activation of platelets and vascular endothelial cells in patients with CHD, evaluation of bleeding liability of these patients, and correlation with the clinical presentation of these patients., Methods: This work was carried out on 20 patients with cyanotic congenital heart diseases (CCHD), 20 patients with acyanotic congenital heart diseases (ACHD), and 20 healthy children who served as the control group, aged between 1 and 10years. All were subjected to full clinical examination, complete blood count, oxygen saturation, echocardiography, bleeding and coagulation times, PT, PTT, FDPs, plasma soluble P-selectin, E-selectin, and platelet factor 4 (PF4)., Results: There was significant prolongation of PT and PTT, and there was a significant lowering of platelet counts. These results were obtained in CCHD and ACHD, but were more significant in CCHD patients. There was a significant elevation in PF4 (55.0±25.5ng/mL), P-selectin (128.9±42.44ng/dL), and E-selectin (9461.5±1701.24pg/mL) levels in children with CCHD as compared to those with ACHD (PF4, 21±7.94ng/mL; P-selectin, 80.1±13.2ng/mL; E-selectin, 7969.6±2127.5pg/mL), and significant increase in both groups when compared to the control group (PF4, 8.1±4.7ng/mL; P-selectin, 27.83±9.73ng/mL; E-selectin, 6750.00±3204.00pg/mL). There was a significant negative correlation between oxygen saturation, plasma P-selectin (r=-0.865), E-selectin (r=-0.401), and PF4 (r=-0.792) in patients with CCHD., Conclusion: Patients with CHD-both cyanotic and acyanotic-have variable degrees of increased liability for both thrombosis and hemorrhage that represents some sort of adaptation to preserve hemostasis and to protect these patients against the clinical presentation of both thrombosis and bleeding. This is to say that CHD patients have their own point of balance between thrombogenicity and bleeding liability. Wide-scale studies are needed to detect the normal levels of different thrombohemorrhagic parameters of these patients., (Published by Elsevier Ltd.)

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from α-granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (α M β 2 , CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by ∼4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the α M I-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a K d of 1.3 ± 0.2 μm Using the PF4-derived peptide library, synthetic peptides duplicating the α M I-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for α M I-domain were identified in the PF4 segments Cys 12 -Ser 26 and Ala 57 -Ser 70 These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Angiogenic activity has been considered to reflect important molecular events during breast tumour development. The present study concerned cellular and molecular changes of MNU-induced breast tumours subjected to promotion and suppression of angiogenesis. Methods: Female Sprague Dawley rats at the age of 21 days received MNU at the dose 70 mg/kg of body weight by intraperitoneal injection. Three months post-carcinogen initiation, mammary tumours were palpated and their growth was monitored. When the tumour diameter reached 1.0 ± 0.05 cm, rats were given bFGF or PF4 intratumourally at a dose of 10 μg/tumour. Entire palpable tumour were subsequently excised and subjected to histology examination, IHC staining, and RT-PCR. Results: No critical morphological changes were observed between pro-angiogenic factor, bFGF, and control groups. However, increase of tumour size with more necrotic and diffuse areas was notable in tumours after anti-angiogenic PF4 intervention. ER and PR mRNA expression was significantly up- and down-regulated in bFGF and PF4 groups, respectively. The trends were significantly associated with peri- and intratumoural MVD counts. However, irrespective of whether we promoted or inhibited angiogenesis, the expression of EGFR and ERBB2 continued to be significantly increased but this was not significantly associated with the MVD score. No significant differences in E-cadherin and LR gene expression were noted between intervention and control groups. Conclusion: ER and PR receptor expression shows consistent responses when tumour angiogenesis is manipulated either positively or negatively. Our study adds to current understanding that not only do we need to target hormonal receptors, as presently practiced, but we also need to target endothelial receptors to successfully treat breast cancer., (Creative Commons Attribution License)

Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, subsets of myeloid cells have also been reported to have anti-tumor properties. It is not clear whether there is a transition between anti- and pro- tumor function of these myeloid cells, and if so, what are the underlying molecular mechanisms. Here we report platelet factor 4 (PF4), or CXCL4, but not the other family members CXCL9, 10, and 11, was produced at higher levels in the normal lung and early stage premetastatic lungs but decreased in later stage lungs. PF4 was mostly produced by Ly6G+CD11b+ myeloid cell subset. Although the number of Ly6G+CD11b+ cells was increased in the premetastatic lungs, the expression level of PF4 in these cells was decreased during the metastatic progression. Deletion of PF4 (PF4 knockout or KO mice) led an increased metastasis suggesting an inhibitory function of PF4. There were two underlying mechanisms: decreased blood vessel integrity in the premetastatic lungs and increased production of hematopoietic stem/progenitor cells (HSCs) and myeloid derived suppressor cells (MDSCs) in tumor-bearing PF4 KO mice. In cancer patients, PF4 expression levels were negatively correlated with tumor stage and positively correlated with patient survival. Our studies suggest that PF4 is a critical anti-tumor factor in the premetastatic site. Our finding of PF4 function in the tumor host provides new insight to the mechanistic understanding of tumor metastasis.

Heparin-induced thrombocytopenia (HIT) is an autoimmune disorder characterised by thrombocytopenia and thrombosis. The mechanisms leading to platelet destruction are complex and the thrombotic complications of HIT appear to be due to multiple different intravascular targets. The dual binding of HIT antibodies to platelet surface PF4/GAG complexes and to FcγRIIA likely leads to both platelet clearance and to their direct activation. Monocytes and endothelial cells bind PF4 with higher avidity than platelets and are more resistant to competitive removal of surface-bound PF4 in the presence of heparin. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface on these cells leads to their activation and increased procoagulant activity. Binding of higher levels of PF4 released from activated platelets to the endothelium may lead to changes of the anticoagulant properties of the glycocalyx and target the endothelial cells for HIT antibodies. Pathogenic antibodies bound to endothelial cells further promote prothrombotic conditions by a mechanism that is independent of FcγR activation, yet not completely understood. A more detailed understanding of the role of monocytes and endothelium may identify new targets for intervention to mitigate the risk of thrombosis with less impact on systemic haemostasis than current approaches to treatment for this serious disorder.

Objective: Causes of profound thrombocytopenia (platelet count <60 K) developing days after cardiac surgery include heparin platelet factor 4 antibodies, thrombotic thrombocytopenic purpura-like antibodies, and endotoxin generated by pulmonary infections. Modulation of immune-mediated profound thrombocytopenia with intravenous immunoglobulin could be efficacious for any of these conditions., Methods: From 2002 to 2010, profound thrombocytopenia developed in 20 consecutive patients within days after cardiac surgery; 19 patients underwent valve or aortic operations, and 1 patient underwent coronary bypass. Risk profiles were high preoperatively: Patients' mean age was 73 years, 50% underwent nonelective procedures, 100% had comorbidities, and 25% underwent reoperations. When decreasing platelet counts approached 60 K, intravenous immunoglobulin was started at 1.5 g/kg intravenously over 5 days. Anticoagulation and platelet transfusions were avoided. In 1 patient, profound thrombocytopenia failed to reverse promptly, and daily plasmapheresis was introduced. Platelet counts before and after interventions were assessed with linear regression analyses over time, including a spline function and statistical knot coincident with starting intravenous immunoglobulin., Results: In 19 of 20 patients, profound thrombocytopenia stabilized and rebounded within 2 to 4 days after initiating intravenous immunoglobulin. In the remaining slow-responding patient, addition of plasmapheresis was associated with rapid recovery. In every patient, coincident multiorgan failure reversed, and 19 of 20 patients recovered uneventfully and survived hospitalization with no limb ischemia or tissue loss. No complications of intravenous immunoglobulin therapy or plasmapheresis were observed., Conclusions: Although mechanisms of profound thrombocytopenia after cardiac surgery are poorly understood, they likely relate to inappropriate autoimmune moieties causing peripheral platelet aggregation and multiorgan failure. A protocol involving immunomodulation with intravenous immunoglobulin supplemented by plasmapheresis appeared safe and efficacious. Direct immunologic interventions for profound thrombocytopenia could improve postoperative outcomes., (Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Many amino acid residue hydrophobicity scales have been created in an effort to better understand and rapidly characterize water-protein interactions based only on protein structure and sequence. There is surprisingly low consistency in the ranking of residue hydrophobicity between scales, and their ability to provide insightful characterization varies substantially across subject proteins. All current scales characterize hydrophobicity based on entire amino acid residue units. We introduce a simple binary but atomic-level hydrophobicity scale that allows for the classification of polar and non-polar moieties within single residues, including backbone atoms. This simple scale is first shown to capture the anticipated hydrophobic character for those whole residues that align in classification among most scales. Examination of a set of protein binding interfaces establishes good agreement between residue-based and atomic-level descriptions of hydrophobicity for five residues, while the remaining residues produce discrepancies. We then show that the atomistic scale properly classifies the hydrophobicity of functionally important regions where residue-based scales fail. To illustrate the utility of the new approach, we show that the atomic-level scale rationalizes the hydration of two hydrophobic pockets and the presence of a void in a third pocket within a single protein and that it appropriately classifies all of the functionally important hydrophilic sites within two otherwise hydrophobic pores. We suggest that an atomic level of detail is, in general, necessary for the reliable depiction of hydrophobicity for all protein surfaces. The present formulation can be implemented simply in a manner no more complex than current residue-based approaches., (© 2013.)

Background: A therapeutic dilemma exists when patients with symptomatic carotid stenosis and concomitant heparin-induced thrombocytopenia (HIT) are advised to urgently undergo carotid endarterectomy (CEA) with heparin therapy., Methods: After a 63-year-old man with HIT and multiple medical comorbidities underwent emergent coronary artery bypass grafting, postoperative imaging revealed plaque at the origin of the left internal carotid artery with 80%-99% stenosis and minimal contralateral internal carotid artery disease. During the patient's evaluation to undergo CEA for symptomatic high-grade carotid stenosis, enzyme-linked immunosorbent assay revealed persistent platelet factor 4 antibodies., Results: The endarterectomy was successfully performed while the patient received argatroban, both as a continuous infusion and intermittent irrigation during dissection of the plaque. Postoperatively, the drip was continued for 24 hours, and the patient was discharged day 2 on a daily dose of 325 mg of aspirin. At the 6-month examination, Doppler ultrasound revealed normal anterograde velocities with no evidence of stenosis, and the patient noted no subsequent ischemic events., Conclusions: We now recommend systemic intravenous and local argatroban irrigation to prevent thromboembolic complications in CEA cases with HIT and renal insufficiency. Bivalirudin for both systemic intravenous use and local irrigation may be safer in patients without renal insufficiency because of its shorter half-life., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Platelet factor 4 (PF4) and tissue factor pathway inhibitor (TFPI) are two proteins with high affinity for heparin. They are each stored in platelets, as well as on endothelial cell surfaces, from where both are displaced or released following an injection of heparin with a rapid and marked increase in serum levels. Prior work has demonstrated that the platelet count is one of the factors affecting the levels of heparin-releasable PF4. We therefore characterized the response to a dose of intravenous heparin previously demonstrated to completely displace PF4 from the non-platelet pool in subjects with normal or increased platelet counts. Seventeen patients with essential thrombocytosis (ET), 10 patients with polycythemia vera and high platelet counts (PV-H), 7 patients with polycythemia vera and normal platelet counts (PV-N) and 10 controls received an initial bolus of 40 I.U./kg of unfractionated heparin, followed 2 hours later by a 2nd bolus of a fixed dose of 1000 I.U. TFPI activity did not show any variation among the different groups, either before (TFPI) or after (HR-TFPI) the first bolus of heparin: ET, TFPI 92.6 ± 21.5%, HR-TFPI 298.3 ± 165.8; PV-H, TFPI 91.5 ± 32.0, HR-TFPI 210 ± 1.0; PV-N, TFPI 69.4 ± 24.0, HR-TFPI 203.0 ± 79.0; C, TFPI 109.5 ± 33.5, HR-TFPI 234.0 ± 60.4. TFPI activity returned to basal values prior to the 2nd injection of heparin, which again elicited a rise in TFPI, albeit smaller due to the lower level of heparin injected. In contrast to the lack of any difference between groups with respect to TFPI, the level of heparin-releasable PF4 (HR-PF4) was significantly higher in ET and PV-H patients compared to PV-N patients or controls. However when normalized for platelet count, both PV-H and PV-N had HR-PF4 levels after the 1st heparin injection that were significantly higher than observed in ET patients (PV-H 1.163 + 0.108, PV-N 1.411 + 0.019, ET 0.737 + 0.086 ng/10/3 platelets) supporting an increased platelet activation in PV. Thus, although platelets contain approximately 5-10% of the total amount of TFPI in plasma, they do not affect the major intravascular pool of TFPI mobilizable by heparin. However, since the concentration at the site of vessel wall injury is enhanced several-fold, TFPI could play a role in competing with PF4 to limit thrombus formation in patients with high platelet count.