Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than α-thrombin

Hackeng, Tilman M.; Tans, Guido; Koppelman, SJ; Groot, Philip G. de; Rosing, Jan; Bouma, Bonno N.

doi:10.1042/bj3190399

Cited by 36 publications

(24 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thrombomodulin expression (ELISA) and activity (ability to activate protein C in the presence of thrombin) was high on non-stimulated HUVEC [130,[135][136][137] and BAEC [132] on TCPS and fibronectin coated TCPS [134] and decreased significantly on activation with an agonist. Thrombomodulin gene expression was also found to be high in HUVEC on TCPS and fibronectin coated TCPS [134,137].…”

Section: Molecular Expression Studiesmentioning

confidence: 99%

The influence of biomaterials on endothelial cell thrombogenicity

2007

View full text Add to dashboard Cite

Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their nonthrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thromobgenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions.

show abstract

Section: Molecular Expression Studiesmentioning

confidence: 99%

The influence of biomaterials on endothelial cell thrombogenicity

2007

View full text Add to dashboard Cite

show abstract

“…Further, the mutant mice failed to show any fibrin deposition in the brain or kidney suggesting that anticoagulation could be regulated differently depending on the tissue (98). On the other hand, in vitro experiments have demonstrated that, in the presence of TM, thrombin and meizothrombin have similar catalytic efficiencies with respect to APC formation (48,100,101). However, in the presence of PCPS vesicles or a cell surface, the catalytic efficiency of meizothrombin for the activation of protein C is 6-fold higher than the activity of thrombin for the same reaction (48,101), and this increased efficiency is specifically due to the binding of meizothrombin to the membrane surface, because meizothrombin desF1 has the same activity as thrombin for the activation of protein C in the presence of TM and in the presence or absence of a membrane surface (48).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, in vitro experiments have demonstrated that, in the presence of TM, thrombin and meizothrombin have similar catalytic efficiencies with respect to APC formation (48,100,101). However, in the presence of PCPS vesicles or a cell surface, the catalytic efficiency of meizothrombin for the activation of protein C is 6-fold higher than the activity of thrombin for the same reaction (48,101), and this increased efficiency is specifically due to the binding of meizothrombin to the membrane surface, because meizothrombin desF1 has the same activity as thrombin for the activation of protein C in the presence of TM and in the presence or absence of a membrane surface (48). Our data show that, in whole plasma, in the absence of TM but in the presence of PCPS vesicles, meizothrombin generates APC much faster than thrombin as shown by the increase in appearance of APC-degradation products from factor Va heavy chain.…”

Section: Discussionmentioning

confidence: 99%

The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

Bukys

Orban

Kim

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

␣-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. ␣-Thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by ␣-thrombin. We have used plasma-derived ␣-thrombin, ␤-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/ R271A) that can only be cleaved at Arg 320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO 3 , and Arg 1689. Cleavage of both procofactors by ␣-thrombin was significantly inhibited by D5Q1,2. In contrast, ␤-thrombin was unable to cleave factor V at Arg 1545 and factor VIII at both Arg 372 and Arg 1689. The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. ␤-Thrombin was found to cleave factor V at Arg 709 and factor VIII at Arg 740 , albeit less efficiently than ␣-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by ␤-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of ␣-thrombin can account for the interaction of both procofactors with ␣-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of ␣-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.Blood clotting involves a multitude of proteins, which act in concert in response to vascular injury to produce the procoagulant enzyme thrombin, which in turn is responsible for the generation of the fibrin plug (1). The two procoagulant enzymatic complexes (i.e. intrinsic tenase and prothrombinase) that are critical for thrombin formation are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. The intrinsic tenase complex is composed of the enzyme, factor IXa, and the cofactor, factor VIIIa, associated on the cell membrane in the presence of divalent metal ions. Similarly, the prothrombinase complex is composed of the enzyme, factor Xa, and the cofactor, factor Va, associated on a cell surface in the presence of divalent metal ions. Although both enzymes factor IXa and factor Xa alone, can activate factor X and prothrombin, r...

show abstract

“…Interestingly, the prothrombin activation product meizothrombin binds to thrombomodulin and is an approximately 6 times more efficient protein C activator than thrombin. 38 Recombinant APC has been approved to treat patients with severe sepsis, although the use remains controversial. 39 It is difficult to predict how degradation of endothelial TFPI by infused APC may affect the outcome in septic patients.…”

Section: Discussionmentioning

confidence: 99%