Dataset Information

Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma.

ABSTRACT: Activated protein C (APC), thrombin, and factor (f) Xa are vitamin K-dependent serine proteases that are key factors in blood coagulation. Moreover, they play important roles in inflammation, apoptosis, fibrosis, angiogenesis, and viral infections. Abnormal activity of these coagulation factors has been related to multiple conditions, such as bleeding and thrombosis, Alzheimer's disease, sepsis, multiple sclerosis, and COVID-19. The individual activities of APC, thrombin, and fXa in coagulation and in various diseases are difficult to establish since these proteases are related and have similar substrate preferences. Therefore, the development of selective chemical tools that enable imaging and discrimination between coagulation factors in biological samples may provide better insight into their roles in various conditions and potentially aid in the establishment of novel diagnostic tests. In our study, we used a large collection of unnatural amino acids, and this enabled us to extensively explore the binding pockets of the enzymes' active sites. Based on the specificity profiles obtained, we designed highly selective substrates, inhibitors, and fluorescent activity-based probes (ABPs) that were used for fast, direct, and simultaneous detection of APC, thrombin, and fXa in human plasma.

SUBMITTER: Modrzycka S

PROVIDER: S-EPMC9200056 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma.

Modrzycka Sylwia S Kołt Sonia S Polderdijk Stéphanie G I SGI Adams Ty E TE Potoczek Stanisław S Huntington James A JA Kasperkiewicz Paulina P Drąg Marcin M

Chemical science 20220427 23

Activated protein C (APC), thrombin, and factor (f) Xa are vitamin K-dependent serine proteases that are key factors in blood coagulation. Moreover, they play important roles in inflammation, apoptosis, fibrosis, angiogenesis, and viral infections. Abnormal activity of these coagulation factors has been related to multiple conditions, such as bleeding and thrombosis, Alzheimer's disease, sepsis, multiple sclerosis, and COVID-19. The individual activities of APC, thrombin, and fXa in coagulation ...[more]

PMID: 35774156

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Project description:Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.

Project description:Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as "type I") kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as "type II") inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.

Dataset Information

Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma.

Publications

Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma.

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