José Hermida

Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation

Uniquely amongst vitamin K‐dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin–thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild‐type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 × 105 m−1·s−1, a dissociation rate constant of 7.61 × 10−2 s−1 and equilibrium binding constant (KD) of 147 nm. It was activated by thrombin over endothelial cells with a Km of 213 nm and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R‐1L, R‐1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased Km (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.

Is EPCR a multi-ligand receptor? Pros and cons.

In the last decade, the endothelial cell protein C/activated protein C receptor (EPCR) has received considerable attention. The role initially attributed to EPCR, i.e. the enhancement of protein C (PC) activation by the thrombin-thrombomodulin complex on the surface of the large vessels, although important, did not go beyond the haemostasis scenario. However, the discovery of the cytoprotective, anti-inflammatory and anti-apoptotic features of the activated PC (APC) and the required involvement of EPCR for APC to exert such actions did place the receptor in a privileged position in the crosstalk between coagulation and inflammation. The last five years have shown that PC/APC are not the only molecules able to interact with EPCR. Factor VII/VIIa (FVII/VIIa) and factor Xa (FXa), two other serine proteases that play a central role in haemostasis and are also involved in signalling processes influencing wound healing, tissue remodelling, inflammation or metastasis, have been reported to bind to EPCR. These observations have paved the way for an exploration of unsuspected new roles for the receptor. This review aims to offer a new image of EPCR in the light of its extended panel of ligands. A brief update of what is known about the APC-evoked EPCR-dependent cell signalling mechanisms is provided, but special care has been taken to assemble all the information available about the interaction of EPCR with FVII/VIIa and FXa.

Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo

The endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested. The ferric chloride carotid artery injury model in mice was chosen to test the effect of anti-EPCR mAb on thrombus formation. The time to total occlusion of the vessel was analysed in three groups, given an isotype control mAb (IC), a blocking (RCR-16) or a non-blocking (RCR-20) anti-EPCR mAb. RCR-16 prevented the interaction between protein C/APC and EPCR as demonstrated by surface plasmon resonance and flow cytometry, and inhibited the activation of protein C on the endothelium. IC and RCR-20 were unable to induce such effects. In vivo , RCR-16 shortened the time to total vessel occlusion with respect to IC [13.4 +/- 1.0 (mean +/- SD) and 17.8 +/- 3.2 minutes, respectively, p<0.001]. Occlusive thrombi lasting for more than one hour were observed in all RCR-16-treated animals, but only in 43% of IC-treated ones. Results with RCR-20 were indistinguishable from those observed with IC. For the first time, a direct relationship between blocking EPCR and thrombosis is demonstrated. Blocking anti-EPCR autoantibodies can predispose to thrombosis episodes and may constitute a new therapeutic target.

The functional properties of a truncated form of endothelial cell protein C receptor generated by alternative splicing

Background A soluble form of endothelial cell protein C receptor (sEPCR) is generated by shedding of the cellular form. sEPCR binds to protein C and factor VIIa and inhibits both the activation of protein C and the activity of activated protein C and factor VIIa. High sEPCR levels may increase the risk of thrombosis.We wanted to explore the possibility of detecting soluble endothelial cell protein C receptor forms generated by alternative splicing. Design and Methods Reverse transcriptase polymerase chain reaction was used to look for new forms of endothelial cell protein C receptor. A yeast expression system was used to generate sufficient amounts of the distinct sEPCR forms. Surface plasmon resonance experiments, chromogenic assays, clotting assays and immunoassays were subsequently performed to characterize a new form of sEPCR that was found. Results We demonstrated, by reverse transcriptase polymerase chain reaction and sequencing, the existence of a new, soluble form of endothelial cell protein C receptor generated by alternative splicing, in which the transmembrane region is replaced by a 56-residue tail (tEPCR). Its cDNA was present in human umbilical vein endothelial cells and in most tissues as well as in lung cancer cells. tEPCR was not located in the membrane of transfected cells.We demonstrated that tEPCR binds to protein C and factor VIIa. tEPCR blocked the generation of activated protein C and inhibited the activity of both activated protein C and factor VIIa. tEPCR was detected, by immunoassays, in the supernatant of lung cancer cells and human umbilical vein endothelial cells. Conclusions A truncated form of alternatively spliced endothelial cell protein C receptor was detected in the endothelium and cancer cells. tEPCR behaves as sEPCR generated by shedding of the cellular endothelial cell protein C receptor.

Limited Ability to Activate Protein C Confers Left Atrial Endocardium A Thrombogenic Phenotype A Role in Cardioembolic Stroke

Background and Purpose— Atrial fibrillation is the most important risk factor for cardioembolic stroke. Thrombi form in the left atrial appendage rather than in the right. The causes of this different thrombogenicity are not well-understood. The goal herein was to compare the activation of the anticoagulant protein C and the thrombomodulin and endothelial protein C receptor/activated protein C receptor expression on the endocardium between right and left atria. Methods— We harvested the atria of 6 monkeys (Macaca fascicularis) and quantified their ability to activate protein C ex vivo and we measured the thrombomodulin and endothelial protein C receptor expression by immunofluorescence. Results— We found the ability to activate protein C decreased by half (P=0.028) and there was lower expression of thrombomodulin in the left atrial endocardium than the right (52.5±19.9 and 72.1±18.8 arbitrary intensity units, mean±standard deviation; P=0.028). No differences were detected in endothelial protein C receptor expression. Conclusions— Impaired protein C activation on the left atrial endocardium attributable to low thrombomodulin expression may explain its higher thrombogenicity and play a role in cardioembolic stroke.

Atrial fibrillation in pigs induces left atrial endocardial transcriptional remodelling

The leading cause of cardioembolic stroke is atrial fibrillation (AF), which predisposes to atrial thrombus formation. Although rheological alterations promote a hypercoagulable environment, as yet undefined factors contribute to thrombogenesis. The role of the endocardium has barely been explored. To approach this topic, rapid atrial pacing (RAP) was applied in four pigs to mimic AF. Left and right endocardial cells were isolated separately and their gene expression pattern was compared with that of four control pigs. The AF-characteristic rhythm disorders and endothelial nitric oxide synthase down-regulation were successfully reproduced, and validated RAP to mimic AF. A change was observed in the transcriptomic endocardial profile after RAP: the expression of 364 genes was significantly altered (p<0.01), 29 of them having passed the B>0 criteria. The left atrial endocardium [325 genes (7 genes, B>0)] was largely responsible for such alterations. Blood coagulation, blood vessel morphogenesis and inflammatory response are among the most significant altered functions, and help to explain the activation of coagulation observed after RAP: D-dimer, 0.49 (1.63) vs. 0.23 (0.24) mg/l [median (interquartile range)] in controls, p=0.02. Furthermore, three genes directly related to thrombotic processes were differentially expressed after RAP: FGL2 [fold change (FC)=0.85; p=0.007], APLP2 (FC=-0.47; p=0.005) and ADAMTS-18 (FC=-0.69; p=0.004). We demonstrate for the first time that AF induces a global expression change in the left atrial endocardium associated with an activation of blood coagulation. The nature of some of the altered functions and genes provides clues to identify new therapeutic targets.

Inducing heat shock protein 70 expression provides a robust antithrombotic effect with minimal bleeding risk

Antithrombotic medications target coagulation factors. Their use is associated with an increased bleeding risk. Safer drugs are needed. The heat shock protein 70 (Hsp70) exhibits antithrombotic properties that do not influence bleeding. By using murine models, we aimed to test the hypothesis that overexpressing Hsp70 with CM-695, a first in class dual inhibitor of HDAC6 and phosphodiesterase 9, protects against thrombosis while leaves bleeding tendency unaltered. CM-695 was used to induce Hsp70 overexpression. Hsp70 overexpressing mice were submitted to three thrombosis-triggering procedures. The ferric chloride carotid artery model was used to compare the antithrombotic role of CM-695 and rivaroxaban, a direct oral anticoagulant. The mouse tail transection model was used to compare the bleeding tendency upon CM-695 or rivaroxaban administration. Intraperitoneal (i. p.) 20 mg/kg CM-695 increased Hsp70 expression markedly in the murine aortic tissue. This treatment delayed thrombosis in the collagen/epinephrine [p=0.04 (Log-Rank test), n=10], Rose Bengal/laser [median vessel occlusion time (OT): 58.6 vs 39.0 minutes (min) in the control group (CG), p=0.008, n≥10] and ferric chloride (OT: 14.7 vs 9.2 min in the CG, p=0.032, n≥10) models. I.p. 80 mg/kg CM-695 (n≥9) and intravenous 3 mg/kg rivaroxaban (n≥8) significantly delayed thrombosis. CM-695 did not induce bleeding [median bleeding time (BT): 8.5 vs 7.5 min in the CG, n≥10]. However, BT was dramatically increased by rivaroxaban (30.0 vs 13.7 min in the CG, p=0.001, n=10). In conclusion, CM-695 is a new antithrombotic small molecule devoid of bleeding risk that may be envisioned as a useful clinical tool.

Insights into venous thromboembolism prevention in hospitalized cancer patients: Lessons from a prospective study

Hospitalized cancer patients are at high risk of venous thromboembolism (VTE). Despite current recommendations in clinical guidelines, thromboprophylaxis with low molecular weight heparin (LMWH) is underused. We performed an observational prospective study to analyse factors influencing prophylaxis use, VTE events and mortality in cancer-hospitalized patients. 1072 consecutive adult cancer patients were included in an University Hospital from April 2014 to February 2017, and followed-up for 30 days after discharge. The rate of LMWH prophylaxis was 67.6% (95% confidence interval [CI] 64.7% to 70.4%), with a 2.8% rate of VTE events (95% CI 1.9% to 3.9%) and 3.5% rate of major bleeding events (95% CI 2.5% to 4.8%). 80% of VTE events occurred despite appropriate thromboprophylaxis. Overall, 30-day mortality rate was 13.2% (95% CI 11.2% to 15.3%). Active chemotherapy treatment, hospital stay ≥ 4 days, and metastatic disease were associated with a higher use of LMWH. On the contrary, patients with hematologic malignancies, anemia or thrombocytopenia were less prone to receive thromboprophylaxis. The main reasons for not prescribing LMWH prophylaxis were thrombocytopenia (23.9%) and active/recent bleeding (21.8%). The PRETEMED score, used for VTE risk stratification, correlated with 30-day mortality. There is room for improvement in thromboprophylaxis use among hospitalized-cancer patients, especially among those with hematologic malignancies. A relevant number of VTE events occurred despite prophylaxis with LMWH. Therefore, identification of risk factors for thromboprophylaxis failure is needed.

Identification of new markers of recurrence in patients with unprovoked deep vein thrombosis by gene expression profiling: the retro study

The aim of this study was to assess differences in the gene expression profile of peripheral blood cells between patients with early recurrent thrombosis vs. patients without recurrent events after withdrawal of anticoagulant therapy for a first episode of unprovoked deep vein thrombosis (uDVT), to identify novel predictors of recurrence.