Complex Interaction between Factor Va-Light Chain and Thrombomodulin in the Regulation of Protein C Activation

SummaryA heterozygous GT transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg-1 to a Leu (PCR-1L) was identified in a thrombophilic patient. The PCR-1L was purified from the patient’s plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PCR-1L revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PCR-1L/propeptide exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PCR-1L/propeptide the propeptide cleavage at Lys-2 is less efficient since approximately 60% of PC variant molecules present in patient’s plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.

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INACTIVATION OF FACTOR VIII BY ACTIVATED PROTEIN C AND PROTEIN S

10.1055/s-0038-1644770 ◽

1987 ◽

Author(s):

P J Fay ◽

S I Chavin ◽

F J Walker

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Heavy Chain ◽

Light Chain ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

Factor Ix ◽

Heavy Chains ◽

Factor Va

Human factor VIII has been isolated from factor VIII concentrates. The isolated protein is composed of a heavy chain and light chain. The heavy chain was heterogenous with respect to molecular weight ranging from 110-170 kDa. The light chain appeared as a 81/84 kDa dimer, 'when factor VIII was treated with activated protein C in the presence of calcium and phospholipids factor VIII procoagulant activity was rapidly lost. Analysis of the activated protein C catalyzed cleavage products of factor VIII indicated that loss of activity was correlated with cleavage of the heavy chains. The heavy chains appeared to be converted into 93 kDa and 53 kDa peptides. A separate factor VIII preparation has been prepared that contained only a 93 kDa heavy chain as well as the 81/83 kDa light chain. When this preparation was inactivated with activated protein C, a pathway in which the 93 kDa peptide was degraded into a 68 kDa peptide which was subsequently degraded into 48 and 23 kDa polypeptides. This result suggested that the 53 kDa polypeptide was not derived from the 93 kDa domain of the heavy chain, but must have been derived from the variable molecular weight portion of the heavy chain. These results suggest that activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. Protein S has been observed to be a protein cofactor both the anticoagulant and proteolytic action of activated protein C with factor Va. It is thought that protein S forms a lipid bound complex with activated protein C which then can rapidly inactivate factor Va. When factor VIII was inactivated in the presence of both activated protein C and protein S the rate of activity loss was enhanced. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products including the 93, 68, and 53 kDa polypeptides. In an analogous reaction, the addition of factor Xa has been observed to inhibit the inactivation of factor Va by activated protein C. The addition of factor IX to the factor Vlll-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Finally, as both factor Va and factor VIII have structural similarities and are substrates for activated protein C the possibility that they might compete as substrates was tested. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.

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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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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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