Hypercoagulable states, also called thrombophilia, can either be congenital or acquired. Congenital thrombophilia, associated mainly with venous thrombosis, is either secondary to coagulation-inhibitor deficiencies, i.e., antithrombin, protein C and Protein S, or gain of function mutations, i.e., factor V Leiden and prothrombin G20210A mutations. Despite the relative frequency of these two mutations, they have not been associated with venous thrombosis recurrence. Most prevalent thrombophilia have a limited impact and usually does not change indications for duration of antithrombotic treatment or prophylaxis compared to decisions based on clinical factors. However, rare inherited thrombophilia such as antithrombin deficiency could justify a long-term anticoagulation. The main acquired thrombophilia, the Antiphospholipid syndrome (APS), is associated with both arterial and venous thrombosis. Its impact on patient management is significant: choice of the anticoagulant (DOAC vs. warfarin), duration of anticoagulation, screening of any organ involvement and systemic autoimmune disease, introduction of immunosuppressive therapy.

BACKGROUND: Protein S (PS) is an anticoagulant that functions as a cofactor for activated protein C (APC) and tissue factor pathway inhibitor. PS deficiency is a risk factor for venous thromboembolism. PS activity is commonly measured using clot-based assays involving fibrin and thrombin production, but improvements are needed.
OBJECTIVE: To develop a new assay for measuring plasma PS activity by quantifying the amount of activated coagulation factor V (FVa) cleaved by APC.
METHODS: We designed a recombinant, modified FV (FVm) that mimicked FVa. We analyzed 160 purposively selected plasma samples from the Biobank of the National Cerebral and Cardiovascular Center.
RESULTS: The assay using mixed normal and PS-deficient plasma detected FVm cleavage in a PS concentration-dependent manner. The correlation between PS activity, measured using the FVm cleavage assay, and free PS antigen levels was relatively weak. We then sequenced all exons of PROS1 from 47 subjects with <60% activity in either the FVm cleavage assay or the clot-based assay. Nonsynonymous variants were identified in 12 of 24 subjects with <60% activity in both assays and in 2 of 7 subjects with <60% activity in the FVm cleavage assay alone. No variants were identified in 16 subjects with <60% activity in the clot-based assay alone. Unlike the clot-based assay, the FVm cleavage assay was not affected by the presence of rivaroxaban in the plasma.
CONCLUSIONS: An assay using the FVm substrate may be less susceptible to interference and provide a more accurate evaluation of plasma PS activity than clot-based assays.

This systematic review and meta-analysis assesses venous thromboembolism (VTE) risk in adults with hereditary thrombophilia, including Factor V Leiden (FVL) mutation, prothrombin G20210A (FII) mutation, compound heterozygosity, protein C (PC), protein S (PS), and antithrombin (AT) deficiency. Eligibility criteria included studies suitable for quantitative synthesis with extractable information on VTE risk in adults (> 15 years). There were no restrictions on VTE type, location, or occurrence. Two authors reviewed all studies and extracted data from 107 publications, encompassing 107,130 individuals (21,560 experiencing VTE). We used a random effects model and calculated odds ratios (ORs) with 95% confidence intervals (CIs). The highest risk was associated with homozygous FVL (OR 5.58, 95% CI 4.61-6.74), homozygous FII (OR 5.16, 95% CI 3.12-8.52), and compound heterozygosity (OR 4.64, 95% CI 2.25-9.58). In contrast, VTE risk was lowest for FVL heterozygosity (OR 2.97, 95% CI 2.41-3.67) and FII heterozygosity (OR 2.21, 95% CI 1.70-2.87), whereas PC (OR 3.23, 95% CI 2.05-5.08), PS (OR 3.01, 95% CI 2.26-4.02), and AT deficiency (OR 4.01, 95% CI 2.50-6.44) demonstrated an intermediate VTE risk. These results highlight an increased risk of venous thromboembolism in adults with hereditary thrombophilia. However, the risk for patients with PC, PS, and AT deficiency appears to be lower than previously stated, likely due to varying thrombogeneity of the underlying genetic mutations. Further research addressing this aspect of VTE risk in hereditary thrombophilia is imperative to improve patient management. TRIAL REGISTRATION: PROSPERO registration number CRD42022376757.

BACKGROUND: Inherited antithrombin, protein C, and protein S deficiency increase the risk of venous thromboembolism. The presence of defects can be identified by clinical laboratory assays. In most Chinese clinical laboratories, the screening tests for antithrombin, protein C, and protein S deficiency are their activity assays. Ensuring appropriate pre-analytical storage conditions for activity tests is essential. This study aimed to assess the effects of storage conditions on antithrombin, protein C, and protein S activity in frozen plasma.
METHODS: We collected the remaining plasma of 29 patients. The baseline of antithrombin, protein C, and protein S activity values were tested within 4 h. Then, each sample was sub-packaged into 4 EP tubes, and was stored at -20 °C for 3 days, -20 °C for 7 days, -80 °C for 3 days, and - 80 °C for 7 days, respectively. After thawing, samples were tested by two systems.
RESULTS: The percentage deviation of antithrombin and protein C activity assay was<10% compared with the initial values. Protein S activity showed a significant reduction in frozen plasma, with a deviation > 10%. Some samples, initially within the normal range, were classified as abnormal after freezing storage.
CONCLUSIONS: Our study indicated that antithrombin and protein C remain stable when stored at -20 °C or -80 °C in a week. We argued that Protein S activity is not stable in frozen plasma. The use of frozen-thawed plasma for PS activity assay may result in overdiagnosis of protein S deficiency.

BACKGROUND: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry.
OBJECTIVE: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19.
METHODS: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD.
RESULTS: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes.
CONCLUSIONS: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2.
CONCLUSIONS: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.

BACKGROUND:  Migraine is associated with several genetic or acquired comorbidities. Studies conducted in recent years emphasize that the frequency of thrombophilia is high in migraine, especially migraine with aura (MA). Similarly, the presence of white matter lesions (WMLs) on brain magnetic resonance imaging (MRI) scans has been associated with migraine for many years.
OBJECTIVE:  Based on the knowledge that both WMLs and thrombophilia variants are frequently observed in MA, we aimed to investigate whether there is a relationship between genetic thrombophilia and the presence of WMLs in these patients.
METHODS:  The levels of proteins S and C, antithrombin III activities, activated protein C (APC) resistance, antiphospholipid immunoglobulin G/immunoglobulin M (IgG/IgM) and anticardiolipin IgG/IgM antibodies were investigated in 66 MA patients between the ages of 18 and 49 years who presented no cardiovascular risk factors. The presence of WMLs and the Fazekas grade was determined from the brain magnetic resonance imaging (MRI) scans\' T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequence taken from the patients. The rates of WMLs were compared in patients with and without thrombophilia.
RESULTS:  Thrombophilia was detected in 34.8% of the patients, and 27.3% were determined to have WMLs in brain MRI scans. The WMLs were detected in 23.3% of the patients without thrombophilia, in 34.8% of those with thrombophilia, and in 50% of the subjects with multiple thrombophilia disorders. Among the thrombophilia disorders, only APC resistance was significantly more common in patients with WMLs.
CONCLUSIONS:  The results of the present study showed that thrombophilia may be a mechanism that should be investigated in the etiology of increased WMLs in MA.
BACKGROUND:  La migraña se asocia con una serie de comorbilidades genéticas o adquiridas. Los estudios realizados en los últimos años destacan que la frecuencia de trombofilia es elevada en la migraña, especialmente en la migraña con aura (MA). De manera similar, la presencia de lesiones de la sustancia blanca (LSB) en las imágenes por resonancia magnética (RM) del cerebro se ha asociado con la migraña hace muchos años.
OBJECTIVE:  Con base en la información de que se suelen observar tanto LSB como variantes de la trombofilia en MA, nuestro objetivo fue investigar si existe una relación entre la trombofilia genética y la presencia de LSB en estos pacientes. MéTODOS:  Se investigaron los niveles de proteína S y de proteína C, actividades de antitrombina III, resistencia a la proteína C activada (PCA), anticuerpos antifosfolípidos inmunoglobulina G/inmunoglobulina M (IgG/IgM) y anticuerpos anticardiolipina IgG/IgM en 66 pacientes con MA entre 18 y 49 años que no presentaban factores de riesgo cardiovascular. Se determinaron la presencia de LSB y el grado de Fazekas a partir de imágenes por RM del cerebro en la secuencia ponderada en T2 y recuperación de la inversión atenuada de fluido (fluid-attenuated inversion recovery, FLAIR, en inglés) obtenidas de los pacientes. Se compararon las tasas de LSB en pacientes con y sin trombofilia.
RESULTS:  Se detectó trombofilia en el 34,8% de los pacientes y LSB en el 27,3%. Las LSB estuvieron presentes en el 23,3% de los pacientes sin trombofilia, en el 34,8% de los que tenían trombofilia, y en el 50% de los que tenían múltiples trastornos trombofílicos. La resistencia a la PCA fue significativamente más común en aquellos pacientes con LSB. CONCLUSIóN:  Los resultados del presente estudio mostraron que la trombofilia puede ser un mecanismo que debe investigarse en la etiología del aumento de LSB en MA.

Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK\'s function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.

Data on the pathophysiological mechanisms of hemostatic alterations in the thrombotic events that occur during Ramadan intermittent fasting (RIF), particularly in the natural coagulation inhibitors, are very limited. Thus, our objective was to evaluate the effect of RIF on the natural anticoagulants level, antithrombin, protein C, and total and free protein S (PS) in healthy participants. Participants were divided into two groups. Group I consisted of 29 healthy fasting participants whose blood samples were taken after 20 days of fasting. Group II included 40 healthy non-fasting participants whose blood samples were taken 2-4 weeks before the month of Ramadan. Coagulation screening tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and plasma fibrinogen level, natural anticoagulants; antithrombin, protein C, free and total PS and C4 binding protein (C4BP) levels were evaluated in the two groups. High levels of total and free PS without change in antithrombin, protein C, and C4BP levels were noted in the fasting group as compared with non-fasting ones (p < 0.05). PT and APTT showed no difference between the two groups. However, the fibrinogen level was higher in the fasting group. In conclusion, RIF was found to be associated with improved anticoagulant activity in healthy participants, which may provide temporal physiological protection against the development of thrombosis in healthy fasting people.

COVID-19 has been associated with alterations in coagulation. Recent reports have shown that protein C and S activities are altered in COVID-19. This may affect the complications and outcome of the disease. However, their exact role in COVID-19 remains uncertain. The aim of the current study was therefore to analyze all papers in the literature on protein C and S activities in COVID-19. We searched three medical electronic databases. Of the 2442 papers, 28 studies were selected for the present meta-analysis. For the meta-analysis, means ± standard deviations with 95% confidence intervals (CI) for protein C and S activities were extracted. Pooled p values were calculated using STATA software. Protein C and S activities were significantly lower in COVID-19 patients than in healthy controls (pooled p values: 0.04 and 0.02, respectively). Similarly, protein C activities were considerably lower in nonsurviving patients (pooled p value = 0.00). There was no association between proteins C or S and thrombosis risk or ICU admission in COVID-19 patients (p value > 0.05). COVID-19 patients may exhibit lower activities of the C and S proteins, which might affect disease outcome; however, additional attention should be given when considering therapeutic strategies for these patients.

OBJECTIVE: Many pieces of literature have reported that inherited and acquired thrombophilia might be a risk factor for recurrent implantation failure (RIF), however, most studies have only focused on RIF patients and not their male partners. We studied the possible association of paternal thrombophilia with RIF risk.
METHODS: Forty-two male partners aged 20-45 suffered from RIF compared with 42 males from couples with at least one successful pregnancy. All participants were investigated for thrombophilia markers.
RESULTS: The prevalence of coagulation Factor V activity was significantly higher in the case group (42.9%) than in the control group (16.7%) (p=0.008) (OR=3.75; 95% CI, 1.38, 10.12). The prevalence of protein C and protein S deficiencies in RIF patients were 4.8% and 2.4%, respectively, and 0% in the controls. The prevalence of antithrombin III (ATIII) deficiency was significantly higher in the case group (19%) than in the control group (2.4%) (p=0.01). None of MTHFR C677T and MTHFR A1298C were statistically significant between the two groups. Combined thrombophilia was 45.2% in the men of the RIF group when compared with the control, 14.2% (p=0.001) (OR = 4.95; 95% CI, 1.75-13.86).
CONCLUSIONS: Paternal thrombophilia may be related to recurrent implantation failure, so evaluation of this factor in RIF patients could be used to identify relevant risk groups and may help in the proper management of these cases to enhance the chance of implantation.