Characterization of a thrombomodulin binding site on protein C and its distinction from an activated protein C binding site for factor Va

Key Points The THBD c.1611C>A mutation (p.Cys537Stop) causes extremely high soluble thrombomodulin levels resulting in trauma-related bleeding. Soluble thrombomodulin acts by enhancing activated protein C generation and by impairing factor Va, factor VIIIa, and thrombin generation.

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Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614856 ◽

1999 ◽

Vol 82 (11) ◽

pp. 1462-1468 ◽

Cited By ~ 22

Author(s):

José Fernández ◽

Jari Petäjä ◽

John Griffin

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Protein C ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor V ◽

Factor Va ◽

Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

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Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649885 ◽

1995 ◽

Vol 74 (04) ◽

pp. 1079-1087 ◽

Cited By ~ 8

Author(s):

Klaus-P Radtke ◽

José A Fernández ◽

Bruno O Villoutreix ◽

Judith S Greengard ◽

John H Griffin

Keyword(s):

Rhesus Monkey ◽

Protein C ◽

Activated Protein C ◽

Pcr Amplification ◽

Amino Acid Sequences ◽

Heparin Binding ◽

Reactive Center ◽

Protein C Inhibitor ◽

Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

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A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648377 ◽

1992 ◽

Vol 67 (01) ◽

pp. 046-049 ◽

Cited By ~ 10

Author(s):

H A Guglielmone ◽

M A Vides

Keyword(s):

Oral Anticoagulant ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

Normal Subjects ◽

Test Sample ◽

Functional Assay ◽

Fast Method ◽

Factor Va ◽

Protein C Activity

SummaryA simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 ± 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p <0.001). Also, a similar correlation (r = 0.93, p <0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

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