Rapid Functional Testing of Protein C with Protac®, a New Protein C Activator (2023)

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They show high sequence similarity to other serine proteases (Stocker et al., 1986). Their insensitivity to endogenous inhibitors qualifies them as a diagnostic tool to identify protein C-related bleeding disorders, for example, the protein C activator Protac® from the southern copperhead snake (Agkistrodon contortrix contortrix) (Gempeler-Messina et al. , 2001; Martinoli and Stocker, 1986). An example of an anticoagulant thrombin inhibitor is the non-enzymatic bonerojaracin type C-like lectin from Bothrops jararaca, which acts in two ways: it inhibits prothrombin activation (Zingali et al., 2001) and thrombin-induced platelet aggregation (Arocas et al. al., 2001). al., 2001). al., 1996).

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Clinically, impairment of the PC anticoagulant pathway has been associated with an increased risk of venous thromboembolism [8-10]. PC deficiency, in which individuals have a congenital or acquired reduction in PC antigen and/or PC activity, is an independent risk factor for thrombosis and can be caused by ex vivo activation of PC by Protac, an activator found in snake venom. identified [11, 12]. ]. However, PC activation in vivo is accelerated by binding of PC to EPCR in the vascular endothelium [4].

When thrombin binds to thrombomodulin (TM), it becomes a potent protein C activator (PC) and an inhibitor of thrombin-activatable fibrinolysis (TAFI). PC activation is enhanced when PC binds to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin production while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of reduced EPCR function on thrombin formation and fibrinolysis, we generated cells that express TM and an EPCR variant (R96C) that does not bind PC.

To determine the impact of EPCR on APC and TAFIa generation and effects on thrombin generation and fibrinolysis, we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type -TM Express . and the EPCR variant R96C (R96C cells).

In the presence of R96C cells, thrombin generation is increased in normal plasma due to decreased PC activation compared to WT cells. Furthermore, in normal plasma, clot lysis is delayed in the presence of R96C cells, although TAFI activation is not enhanced. In PC-deficient plasma, clot lysis is delayed in the presence of WT and R96C cells due to increased TAFI activation.

We show that impaired EPCR function can be detected by thrombin formation and clot lysis assays in cells expressing TM and EPCR. We have also shown that EPCR deficiency has procoagulant effects, resulting in a delay in clot lysis.

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The conversion of protein C to activated protein C (APC) is important because APC regulates the rate of clotting by inactivating factors VIIIa and Va. Protein C activators have been isolated from many viper species [27], the best studied form being the North American copperhead Agkistrodon contortrix contortrix, from which the Protac® diagnostic tool was developed [28]. Protac® allows the determination of Protein C by conversion to APC, whose activity can be determined by activated partial thromboplastin time or using a chromogenic assay.

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The mixture was then incubated at 37°C for 4 minutes. After 3 minutes, 0.025 mol/L of CaCl2 (100 μL) was added and APC levels were quantified blindly using the activated partial thromboplastin time measurement method.14 Activated PC levels correlated with time of activated partial thromboplastin in standard plasma.

Circulating markers indicative of atherosclerotic plaque instability may have diagnostic and prognostic value in patients with acute coronary syndromes. We evaluated activated protein C (APC), which has antithrombotic, anti-inflammatory, and profibrinolytic properties, as a potential clinical predictor of ST-segment elevation myocardial infarction (STEMI), including return of spontaneous circulation after sudden cardiac arrest.

Patients with STEMI whose APC was measured on arrival at the emergency department were included in this study (n = 335). The primary endpoint was in-hospital death from any cause.

The APC value ranged from 29% to 142%, with a median of 80%. The unadjusted mortality rate progressively increased with decreasing APC values ​​(33.7% in the 1st quartile, 12.7% in the 2nd quartile, 6.0% in the 3rd quartile and 3.6% in the 4th quartile).P< 0.001). This association remained significant in subgroups of patients with STEMI alone (P= 0.04) or with spontaneous cycle return (P= 0.01). After adjusting for independent predictors of in-hospital death, the odds ratio of death among those in the first quartile of APC scores was 9.4 (95% CI 1.1-81.6).P= 0.04). An APC cutoff value of 65% had the highest combined sensitivity and specificity in predicting death.

Measuring APC levels provides predictive information for use in risk stratification across the STEMI spectrum. Decreased APC levels may be a consistent feature in patients at high risk of dying after STEMI.

Prehospital resuscitation with crystalloids exacerbates fibrinolysis, which is associated with high mortality. We hypothesize that, compared to crystalloid resuscitation, plasma prevents hyperfibrinolysis in an environment rich in tissue plasminogen activator (tPA), preserving essential proteins for the regulation of fibrinolysis.

Healthy subjects donated blood that was tested using native (non-activated) thrombolastography (TEG). Whole blood was mixed in progressive dilutions with normal saline (NS) or low platelet plasma (PPP). Tissue plasminogen activator was added to promote a fibrinolytic environment. In a separate experiment, PPP was passed through a 100 kDa filter and the remaining liquid at the top of the filter (TFP) and below the filter (BFP) was obtained. Whole blood was diluted to 50% with TFP, BFP and NS and tested with a tPA-TEG challenge. TFP and BFP were tested for protein concentration and protein composition.

Normal saline and PPP dilution of whole blood without tPA had no effect on clot lysis at 30 minutes (LY30) (NS Spearman's rho 0.300, p=0.186 and PPP 0.294, p=0.288). The addition of tPA increased the NS dilution percentage of LY30 whole blood (0.844, p<0.001), but not significantly (0.270, p=0.202) with PPP dilution. The difference in LY30 between whole blood and PPP-diluted whole blood (mean change -1.05, 95% CI, -9.42 to 7.33) was similar to that for TFP (1.23, 95% CI, -5 .20 to 7.66, p=0.992). However, both BFP (37.65, 95% CI 24.47 to 50.82, p=0.001) and NS (47.36, 95% CI 34.3 to 60.45, p <0.001) showed an increase accentuated in fibrinolysis compared to PPP.

Dilution of crystalloids and whole blood plasma does not enhance fibrinolysis. However, NS dilution of whole blood increases susceptibility to tPA-mediated fibrinolysis. Plasma resuscitation, simulated by plasma dilution of whole blood, reduces the increased susceptibility to tPA-mediated fibrinolysis. The advantages of plasma resuscitation lie in the preservation of plasma proteins.

A family history (FH) of Alzheimer's disease (AD) affects mitochondrial function and may modulate the action of the 40 kDa mitochondrial outer membrane translocase (TOMM40) rs10524523 ('523) amnesic poly-T length.

For 912 middle-aged and 365 middle-aged adults on the AD spectrum, mixed linear models measured HF andTOMM40'523 Interactions in memory and global cognition between baseline and up to 10 years later. A biomarker of CSF mitochondrial function was also evaluated.

In FH-negative participants, preservation of memory and global cognition per gene dose was observed in 'very long' and 'short' carriers. In FH positives, an opposite decrease in gene dose was seen in very long carriers compared to short carriers. Maternal FH was a stronger predictor in middle-aged participants but not in middle-aged participants. Similar gene dose effects were observed for the mitochondrial biomarker aspartate aminotransferase.

These results may clarify conflicting resultsTOMM40Poly-T length and AD-related decline.

Antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss caused by pathogenic antiphospholipid antibodies (aPLA). We have recently shown that Toll-like receptor 2 (TLR2) and CD14 contribute to the activation of aPLA monocytes.

Study the mechanisms of cell activation by aPLA, which leads to procoagulant and proinflammatory responses.

For this study, we used antibodies purified from the plasmas of 10 different patients with APS and healthy donors. We show that aPLA, but not control IgG, is located on human monocytes along with TLR2 and TLR1 or TLR6. Blocking antibodies against TLR2, TLR1, or TLR6, but not against TLR4, reduced the TNF and tissue factor (TF) response to aPLA. Pharmacological and siRNA approaches revealed the importance of clathrin/dynamin-dependent endocytic signaling in cellular activation by aPLA. Furthermore, soluble aPLA induced NF-κB activation, whereas aPLA granules that were not internalized did not activate NF-κB. The internalization of aPLA in monocytes and the activation of NF-κB were dependent on the presence of CD14.

We show that TLR2 and its co-receptors TLR1 and TLR6 contribute to the pathogenicity of aPLA, that aPLA is internalized via clathrin- and CD14-dependent endocytosis, and that endocytosis is required for NF-κB activation. Our results contribute to a better understanding of APS and offer a possible therapeutic approach.

Routine drug monitoring is not required for the two new direct factor Xa inhibitors, apixaban and rivaroxaban. Quickly available test results can be helpful in case of bleeding or before emergency surgery.

The aim of this study was to evaluate the applicability of two modifications of rotational thrombus elastometry (ROTEM®): ROTEM® activated by low tissue factor (LowTF-ROTEM®) and ROTEM® activated by prothrombinase-induced clotting time (PiCT®-ROTEM). . ® ) for determination of apixaban and rivaroxaban in vitro and ex vivo.

Blood samples from 20 volunteers were spiked with apixaban/rivaroxaban to obtain samples with increasing drug concentrations ranging from 50-400 ng/mL. ROTEM® assays modified with LowTF and PiCT® and the determination of the corresponding anti-factor Xa activity were performed in duplicate on 280 samples. The LowTF-ROTEM® test was performed on samples from 20 patients on apixaban or rivaroxaban therapy and 20 control subjects.

There was a strong correlation between apixaban/rivaroxaban plasma concentrations and the parameters LowTF-ROTEM® time to clot (CT; Spearman correlation coefficient (SCC) 0.81 and 0.81, respectively) and time to maximum velocity (t , MaxVel; SCC: 0.81 and 0.80, respectively). ) and a low to moderate correlation for the PiCT®-ROTEM® CT parameters (SCC: 0.38 and 0.59, respectively) and t,MaxVel. (0.51 and 0.69, respectively) in in vitro experiments. LowTF-ROTEM-CT was significantly longer in patients receiving apxiaban or rivaroxaban therapy compared to controls.

LowTF–ROTEM® can be a valuable diagnostic tool to quickly determine the effect of apixaban and rivaroxaban at the point of care.