Page 189 - Haematologica May 2022

Letters to the Editor
Impaired in vivo activated protein C response rates indicate a thrombophilic phenotype in inherited thrombophilia
Venous thromboembolism (VTE) is a multifactorial dis- ease. Hereditary risk factors include the common muta- tions factor V Leiden (FVL) and prothrombin (FII) 20210G>A, with a prevalence of 3–15% among whites, as well as deficiencies of the coagulation inhibitors antithrombin (AT), protein C (PC), and protein S.1 In the recent past, novel risk loci have been found by genome- wide association studies.2,3 However, their consideration in addition to the classical thrombophilic defects results in an estimated heritability of VTE of only 15%, in con- trast to 40–60% heritability observed in family-based studies.4 In order to identify further unknown genetic thrombophilic defects, consideration of the laboratory phenotype of increased thrombin formation in addition to the clinical phenotype of VTE has been proposed, based on the observation of elevated in vitro thrombin generation parameters in families with unexplained thrombophilia and in carriers of genetic variations in hemostasis-related genes other than FVL and FII
20210G>A.5
It remains unclear, however, if increased in vitro throm-
bin formation rates indeed reflect increased in vivo throm- bin formation. In order to investigate this, we compara- tively analyzed in vitro and in vivo thrombin formation in a cohort of healthy individuals and in thrombophilic patients. In vivo coagulation activation was induced by low-dose recombinant activated factor VII (rFVIIa). Subsequent hemostasis biomarker-monitoring included measurement of activated PC (APC) as a measure of the endothelial-dependent anticoagulant response. Recently, using this stimulated hemostasis activity pattern evalua- tion (SHAPE) approach, we were able to show increased in vivo thrombin generation rates and a comparable APC response in FVL and FII 20210G>A carriers.6,7 Moreover, we found that APC response rates correlated with the thrombotic risk in FVL carriers.7
The study population consisted of 30 healthy individu- als and 51 patients with a history of VTE, thereof 28 FVL or FII 20210G>A carriers (FVL/FII 20210G>A cohort), and 23 unrelated subjects with unexplained familial VTE (FH cohort). A diagram of patient recruitment and selec- tion criteria is shown in Figure 1, along with a description of study procedures. Blood samples were drawn before
Figure 1. Eligibility criteria and study procedures. Healthy individuals were recruited from blood donors. Patients with a history of venous thromboembolism (VTE) were recruited from the thrombophilia outpatient clinic of our hospital. The study proposal was approved by the Ethics Committee of the Medical Faculty of the University Bonn (reference number 016/16). Written informed consent was received prior to participation. All finally included study participants (n=81) received morning administration of 15 μg/kg recombinant activated factor VII (rFVIIa) as single intravenous bolus injection after overnight fast. Blood samples were drawn immediately before and 10 minutes (min), 30 min, 1, 2, 3, 5, and 8 hours after administration, each from a new venipuncture. After discarding the first 2 mL, blood was drawn into citrate tubes (10.5 mmol/L, Sarstedt, Nümbrecht, DE). Citrate tubes were supplemented with aprotinin (10 μmol/L) and bivalirudin (250 μg/mL) for activated protein C (APC) measurement. Plasma samples were obtained by centrifugation (2,600 x g, 10 min) within 30 min and stored at less than -70°C until assayed. All finally included study participants completed rFVIIa administration and follow-up blood sampling. All collected sam- ples were analyzed. *Surgery, trauma, immobilization, pregnancy, and puerperium. #Transaminases, g-glutamyl transferase, urea, creatinine in serum. **Decreased plasma levels of antithrombin, protein C, protein S, anti-cardiolipin and anti-β2 glycoprotein I immunoglobulin G (IgG) and IgM, functional lupus anti- coagulants (activated partial thromboplastin time, dilute Russell viper venom time), and factor V Leiden (FVL) and prothrombin (FII) 20210G>A mutation (except for inclusion into the FVL/FII 20210G>A cohort).
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