Matrix metalloproteinase-10 is upregulated by thrombin in endothelial cells and increased in patients with enhanced thrombin generation.

OBJECTIVE: Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. METHODS AND RESULTS: Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. CONCLUSIONS: Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

In vitro hypercoagulability and ongoing in vivo activation of coagulation and fibrinolysis in COVID-19 patients on anticoagulation.

BACKGROUND: COVID-19 is associated with a substantial risk of venous thrombotic events, even in the presence of adequate thromboprophylactic therapy. OBJECTIVES: We aimed to better characterize the hypercoagulable state of COVID-19 patients in patients receiving anticoagulant therapy. METHODS: We took plasma samples of 23 patients with COVID-19 who were on prophylactic or intensified anticoagulant therapy. Twenty healthy volunteers were included to establish reference ranges. RESULTS: COVID-19 patients had a mildly prolonged prothrombin time, high von Willebrand factor levels and low ADAMTS13 activity. Most rotational thromboelastometry parameters were normal, with a hypercoagulable maximum clot firmness in part of the patients. Despite detectable anti-activated factor X activity in the majority of patients, ex vivo thrombin generation was normal, and in vivo thrombin generation elevated as evidenced by elevated levels of thrombin-antithrombin complexes and D-dimers. Plasma levels of activated factor VII were lower in patients, and levels of the platelet activation marker soluble CD40 ligand were similar in patients and controls. Plasmin-antiplasmin complex levels were also increased in patients despite an in vitro hypofibrinolytic profile. CONCLUSIONS: COVID-19 patients are characterized by normal in vitro thrombin generation and enhanced clot formation and decreased fibrinolytic potential despite the presence of heparin in the sample. Anticoagulated COVID-19 patients have persistent in vivo activation of coagulation and fibrinolysis, but no evidence of excessive platelet activation. Ongoing activation of coagulation despite normal to intensified anticoagulant therapy indicates studies on alternative antithrombotic strategies are urgently required.

Identification of 58 Mutations (26 Novel) in 94 of 109 Symptomatic Spanish Probands with Protein C Deficiency.

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene (PROC) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range.

Impaired factor XIIa-dependent activation of fibrinolysis in treated antiphospholipid syndrome gestations developing late-pregnancy complications.

OBJECTIVE: The objective of the study was to investigate the potential role of impaired factor XII-dependent activation of fibrinolysis in treated antiphospholipid syndrome gestations developing late-pregnancy complications. STUDY DESIGN: This was a prospective study in a third-level teaching hospital, including 75 patients: 25 pregnant patients having the antiphospholipid syndrome and carrying their pregnancies until 26 weeks' gestation or later (group 1); 25 pregnant patients having normal term pregnancies and delivery and no previous miscarriage (group 2); and 25 pregnant patients being diagnosed as having severe pre-eclampsia and/or intrauterine growth restriction but testing negative for antiphospholipid antibodies (group 3). Hemostatic evaluation was carried out from patients in groups 1 and 2 between 6 and 10 weeks, between 18 and 22 weeks, and between 28 and 32 weeks' gestation. Patients in group 3 were sampled between 28 and 32 weeks. An additional blood sample was obtained 4 to 6 months after delivery (baseline). The Mann-Whitney U test, the Friedman test, and the chi2 test were used. RESULTS: Patients in group 1 were characterized by increased factor VIIa levels, increased prothrombin fragment 1+2 levels, reduced factor XIIa levels, diminished functional urokinase-type plasminogen activator levels, and decreased levels of plasmin/alpha-2-plasmin inhibitor complexes. These abnormalities were more evident in patients in group 1 developing pre-eclampsia and/or intrauterine growth restriction. CONCLUSIONS: Impaired factor XIIa-dependent activation of fibrinolysis seems to be a key mechanism related to late-pregnancy complications in patients with the antiphospholipid syndrome.

Prevalence of thrombophilia in women with severe ovarian hyperstimulation syndrome and cost-effectiveness of screening.

OBJECTIVE: To determine the prevalence of markers of thrombophilia in patients with severe ovarian hyperstimulation syndrome (OHSS) and to evaluate the cost-effectiveness of screening for factor V Leiden and prothrombin G20210A mutations in women entering an IVF program. DESIGN: Case-control study and cost-effectiveness analysis. SETTING: University teaching hospital. PATIENT(S): Women undergoing controlled ovarian hyperstimulation for IVF complicated by severe OHSS (group 1, n = 20), women undergoing controlled ovarian hyperstimulation for IVF without development of severe OHSS (group 2, n = 40), and healthy control subjects (group 3, n = 100). INTERVENTION(S): Investigation of markers of thrombophilia. Estimate of number of IVF patients needed to detect a case of severe OHSS and thrombosis associated with thrombophilia genetic mutation was calculated from the available data. MAIN OUTCOME MEASURE(S): Blood samples were analyzed for inherited (resistance to activated protein C due to the factor V Leiden mutation; prothrombin G20210A mutation; deficiencies in antithrombin, protein C, and protein S) and acquired (presence of circulating lupus anticoagulants and/or anticardiolipin antibodies; deficiencies of antithrombin and protein S; acquired protein C resistance) markers of thrombophilia. The cost of preventing one thrombotic event in a patient developing severe OHSS after IVF and having factor V Leiden or prothrombin G20210A mutations was calculated. RESULT(S): None of the OHSS patients or controls had antithrombin, protein C, or free protein S deficiencies. All of them tested negative for antiphospholipid antibodies. No patient in group 1 had the factor V Leiden or prothrombin G20210A mutations. The prothrombin G20210A mutation was detected in 1 out of 40 patients (2.5%) in group 2. Both factor V Leiden and prothrombin G20210A mutations were detected in two of the control subjects (2%) (group 3). The estimated cost of preventing one thrombotic event arising as a consequence of screening for factor V Leiden and prothrombin G20210A mutation is a minimum of 418,970 dollars and 2,430,000 dollars, respectively. CONCLUSION(S): The prevalence of thrombophilia is not increased in women with severe OHSS. Screening for V Leiden and prothrombin G20210A mutation in an IVF general population is not cost-effective.